巴西专利BR112015030041B1 culture chamber and culture method

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CHAMBER OF CULTURE AND CULTURE METHOD. The present invention relates to a culture chamber that can prepare uniformly sized spheroids with high efficiency and provided with a microspace structure that is designed to facilitate the replacement of a medium and the collection of cells. The culture chamber includes a plurality of recesses (10) formed in each of a bottom portion (11) and an opening portion (12). The bottom portion (11) has one of a hemispherical shape and a truncated cone shape and the opening portion (12) is defined by a wall that surrounds an area of a boundary between the opening portion (12) and the portion bottom (11) to an end of each of the recesses (10), where the wall has a taper angle in a range of 1 degree to 20 degrees. An equivalent diameter of the limit is in a range of 50 () m to 2 mm and a depth from the bottom of the bottom portion (11) to the end of each of the recesses is in a range of 0.6 or more times 3 or less times the equivalent diameter, and the wall that defines the opening portion (12) forms a continuous surface to (...).
公开号:BR112015030041B1
申请号:R112015030041-3
申请日:2014-06-05
公开日:2021-01-12
发明作者:Satoru Ayano；Naoto Fukuhara；Takanori Takebe；Yoko Ejiri；Hideki Taniguchi
申请人:Kuraray Co., Ltd.；Public University Corporation Yokohama City University；
IPC主号:

专利说明:

Technical Field
[0001] The present invention relates to cell culture and cell collection. Background of the Invention
[0002] Along with the recent development of cell technology, new culture methods are being developed to obtain cells that have a function similar to an in-vivo function by imitating an in-vivo environment or pericellular morphology. An attempt was made to use cells cultured by methods as a simulator for biological treatment or reaction. Various culture methods have been developed, such as a cell culture method when using a culture medium made up of a sponge or fiber; a suspension culture method in which cells are suspended in a medium so that the cells spontaneously form a spheroid; and a method of culturing cells to form a spheroid when performing a non-adherence treatment of cells in a conventional culture chamber (a flask or the like). In particular, a spheroid culture is an excellent method by which cell interactions can be maintained, and the method is thus applied to various cells such as pancreatic islet cells, liver cells, stem cells, and cancer cells. In recent years, studies have been done that focus on the size of a spheroid. For example, in a drug selection test using cancer cells, the diameter or volume of a spheroid is used as an index (Non-Patent Literature 1). It is also reported that cells have different functions depending on the size of a spheroid (Literature other than Patent 2 and 3). In addition to the spheroid formation technique as mentioned above, a spheroid size control technique has attracted attention. In addition, since it is possible to reproduce a specific function of a cell, it is expected that this technique of controlling the size of a spheroid is applicable in several fields, for example, in the fields of artificial organs and bioreactor. In such applications, a technique for preparing a large number of spheroids and recovering spheroids is important.
[0003] As a means to create a spheroid that has a uniform diameter, Patent Literature 1 discloses a method of controlling the size of each spheroid formed by changing the number of cells to be seeded in a 96WP with a shaped background of V on which a hydrophilic membrane is formed. However, the number of spheroids per culture area is small and, therefore, it is difficult to prepare a large number of spheroids. Like other methods for creating a spheroid that has a uniform diameter, Patent Literature 2 to 4 disclose methods of forming a spheroid in a microspace. List of Citations Patent Literature
[0004] [Patent Literature 1] Unexamined Japanese Patent Application Publication No. H08-131153
[0005] [Patent Literature 2] Unexamined Japanese Patent Application Publication No. 2010-88347
[0006] [Patent Literature 3] Publication of International Patent Application No. WO 2012/036011
[0007] [Patent Literature 4] Publication of International Patent Application No. WO 2013/042360 Non-Patent Literature
[0008] [Non-Patent Literature] Juergen Friedrichl, et al., "Spheroid-based drug screen: considerations and practical approach", PROTOCOL, February 12, 2009 (published online) pages 309 to 324
[0009] [Non-Patent Literature 2] Franziska Hirschhaeuser of the patent, et al., "Multicellular tumor spheroids: An underestimated tool is catching up again", Journal of Biotechnology 148, 2010, pages 3 to 15
[0010] [Non-Patent Literature 3] C'ELINE LIU BAUWENS, et al., "Control of Human Embryonic Stem Cell Colony and Aggregate Size Heterogeneity Influences Differentiation Trajectories", STEL CELL, 2008, pages 2300 to 2310. Detailed Description of the Invention Technical Problem
[0011] However, in the culture method disclosed in Patent Literature 1, the efficiency of the culture is extremely low, which constitutes a rate limiting step for large-scale culture. In the culture methods disclosed in Patent Literature 2 and 3, the efficiency of spheroid formation per unit area is high, but there is a possibility that spheroids are removed from the interior of the culture space during medium replacement. Therefore, careful attention is required when changing the medium. In addition, a study was done on a method of causing a part of a spheroid to adhere to the inside of a microspace in order to prevent removal of the spheroid (Patent Literature 4). However, since the adhesion property is different in each cell type, it is necessary to consider a method of surface treatment for each of the cells to be used and, therefore, the method is impractical.
[0012] The present invention was developed in view of the above mentioned background. An objective of the present invention is to design a microspace structure that facilitates the replacement of a medium and the collection of cells, and to provide a culture chamber that has the said microspace structure, and a culture method that uses said chamber culture, to make possible the preparation of uniformly sized spheres with high efficiency, or the preparation of a large number of uniformly sized spheres with high efficiency. Solution to the Problem
[0013] According to one aspect of the present invention, a culture chamber according to one embodiment includes a plurality of recesses, each of which is formed by a bottom portion and an opening portion. The bottom portion is shaped between a hemispherical shape and a truncated cone shape. The opening portion is defined by a wall that surrounds an area of a boundary between the opening portion and the bottom portion to an end of each of the recesses, where the wall has a tapering angle in a 1 degree range at 20 degrees. In addition, an equivalent diameter of the limit is in a range of 50 μm to 2 mm and a depth from the bottom of the bottom portion to the end of each of the recesses is in a range of 0.6 or more times to 3 or least times the equivalent diameter. The wall defining the opening portion forms a continuous surface for the bottom portion, and an inclination of the continuous surface changes at the limit.
[0014] In the culture chamber according to a modality, it is preferable that the end of each recess has a shape between a hemispherical shape, a trapezoidal shape and an inverted triangular shape. It is also preferable that an area between two adjacent recesses is flat and a distance between the two recesses is in the range of 5 μm to 50 μm.
[0015] Furthermore, in the culture chamber according to a modality, it is preferable that the culture chamber is a resin mold formed by one or a combination of two or more selected from the group consisting of acrylic resin, polylactic acid , polyglycolic acid, styrene resin, acrylic styrene copolymer resin, polycarbonate resin, polyester resin, polyvinyl alcohol resin, ethylene copolymer resin and vinyl alcohol, thermoplastic elastomer, vinyl chloride resin and resin silicon. It is preferable that a functional group is formed in the recesses by a method of treating surface modification of any one of the plasma treatment, the coating with glass, the luminous electrical discharge and the ozonation with UV, or a combination thereof, and that the treatment is carried out in such a way that the water contact angle is 45 degrees or less.
[0016] It is preferable that a hydrophilic polymer chain that inhibits cell adhesion is immobilized in the recesses.
[0017] It is preferable that a phospholipid or a phospholipid and polymer complex is immobilized in the recesses.
[0018] It is preferable that each of the recesses have a cell non-stick surface where at least one polymer of a hydrophilic polymer chain that inhibits cell adhesion, and a phospholipid, or a phospholipid and polymer complex, is immobilized after a functional group has been formed in the recesses by a method of treating surface modification of any of the plasma treatment, glass coating, luminous electrical discharge and UV ozonation, or a combination thereof, and that the treatment is carried out so that the angle of contact of the water is 45 degrees or less.
[0019] It is preferable that the hydrophilic polymer chain is poly (hydroxyethyl) methacrylate, and it is more preferable that an average molecular weight of poly (hydroxyethyl) methacrylate is 100,000 or more.
[0020] In accordance with one aspect of the present invention, a culture method according to one embodiment uses any of the culture chambers described above. This culture method includes: the dispersion of cells in a medium, where a total number of cells is equal to or greater than a number (N) of the recesses of the culture chamber and equal to or less than a number obtained by multiplying the number (N) of the recesses by a value obtained by dividing a volume (V1) of a space defined by each of the recesses by a volume (V2) of the cells to be seeded; and adding the medium to the culture chamber.
[0021] In one aspect of the culture method according to one embodiment of the present invention, it is preferable that a spheroid is formed in a space defined by each of the recesses, and it is more preferable that a spheroid is formed in space and the spheroid can grow (proliferate).
[0022] In the case of differentiating and inducing a spheroid, the spheroid is preferably induced in a state in which the spheroid is formed in space.
[0023] It is preferable that 60% or more of a total number of spheroids formed in the culture chamber have a diameter in a range of ± 5% of an average spheroid diameter.
[0024] It is preferable that the cells in the recesses are recovered by shaking the medium, and it is more preferable that the shaking of the medium is done by any of the following means: shaking the medium when shaking the culture chamber; agitation of the medium when sucking and discharging the medium; stirring the medium by arranging a stirring paddle in the culture chamber; and shaking the medium by placing a stirrer in the culture chamber, or a combination thereof.
[0025] It is preferable that the medium is replaced at least once and that 20% or more of the medium is replaced.
[0026] According to another aspect of the present invention, a culture method according to one embodiment uses any of the culture chambers described above. The culture method for sowing the cells, culturing the cells, replacing a medium and collecting the cells includes the steps of: a) dispersing the cells in a medium, where the number of cells is equal to or greater than a number (n) of the recesses in the culture chamber is equal to or less than a number obtained by multiplying the number (n) of the recesses by a value obtained by dividing a volume (V) of each of the recesses by a volume (v ) of the cells to be seeded, and the addition of the medium to the culture chamber; b) culturing the cells in the culture chamber for 12 hours or more to form a spheroid; c) suction of 20% or more of the medium and then the injection of the same amount of fresh medium; d) repeating steps a) to c) a plurality of times to allow the spheroid to grow; e) placing the spheroid to grow to a desired size and then shaking the medium to suspend the cells within the recesses in the medium; and f) suction of the medium including the cells by a suction machine to recover the cells. Advantageous Effects of the Invention
[0027] According to the present invention, it is possible to provide a culture chamber with the capacity to prepare a large number of spheres with a uniform size with high efficiency and to have a microspace structure that is designed to allow the replacement of a medium and the collection of cells, and a culture method that uses the culture chamber. Brief Description of Drawings
[0028] Figure 1 is a diagram showing an example of a culture chamber according to a modality;
[0029] Figure 2 is a cross-sectional view showing an example of the shape of a recess according to a first embodiment;
[0030] Figure 3 is a top view showing an example of the shape of the recess according to the first embodiment;
[0031] Figure 4 is a diagram showing an example of the shape of a recess formed when using a part of a spherical shape according to a second modality;
[0032] Figure 5 is a diagram showing another example of the shape of a recess formed when using a part of a spherical shape according to the second modality;
[0033] Figure 6 is a diagram showing an example of the shape of a recess formed when using a truncated cone shape according to the second modality;
[0034] Figure 7 is a diagram showing another example of the shape of a recess according to the second modality;
[0035] Figure 8 is a diagram showing an example of the format of an opening portion according to a third embodiment;
[0036] Figure 9 is a diagram showing another example of the shape of an opening portion according to the third embodiment;
[0037] Figure 10 is a diagram showing an example of the structure of a culture chamber according to a fourth embodiment;
[0038] Figure 11 is a diagram showing another example of the culture chamber structure according to the fourth modality;
[0039] Figure 12 is a diagram showing yet another example of the culture chamber structure according to the fourth modality;
[0040] Figure 13 is a diagram showing a spheroid overexperience rate in one example and a spheroid survival rate in a comparative example when replacing a medium;
[0041] Figure 14 shows a photograph illustrating an image of the spheroids in the example and a photograph illustrating an image of the spheroids in the comparative example before and after replacing a medium;
[0042] Figure 15 shows a photograph that illustrates an image of the cells before the cells are recovered in the example and a photograph that illustrates an image of the cells after the cells are recovered in the example; and
[0043] Figure 16 shows photographs of the spheroids recovered from a culture chamber according to the example. Description of Modalities
[0044] The modalities will be described below with reference to the drawings. To clarify the explanation, omissions and simplifications are made as necessary in the following description and in the drawings. Throughout the drawings, the constituent elements have the same configuration or function, or the corresponding parts are denoted by the same reference numerals, and their descriptions are omitted. First modality <Chamber of Culture>
[0045] Figure 1 is a diagram showing an example of a culture chamber according to a modality. Figure 1 shows a part of a culture plate 3 that includes a plurality of culture chambers 1. Figure 1 shows a part of a culture plate 3 that includes a plurality of culture chambers 1. The upper part of Figure 1 shows some recesses of a plurality of recesses 10 which are formed at the bottom of each of the culture chambers 1, when viewed from the top of the culture plate 3. The plurality of recesses 10 is arranged in each of the culture chambers 1. In In terms of the production of the culture chambers 1 and the efficiency of the cell culture, it is preferable to arrange the plurality of recesses in a regular manner. A culture chamber 1 corresponds, for example, to a well arranged in a plate that includes a plurality of wells. In other words, the plurality of recesses 10 is arranged in the respective cavities of a plate with cavities.
[0046] A well plate is an experimental / test instrument formed from a flat plate that has a number of notches (holes or cavities), and each well is used as a test tube or petri dish . The number of wells is, for example, 6, 24, 96, 384 or more. Examples of the shape of the bottom of each well include a flat shape, a round shape and a combination of a number of elongated microtubes (plate with deep cavities).
[0047] Each recess 10 forms a microspace, which is a small space for cell culture and, therefore, each recess can also be indicated as a microchamber.
[0048] Figures 2 and 3 show an example of the shape of a recess according to a first modality. Figure 2 shows a cross-sectional view of a recess 10, and Figure 3 shows a top view of a recess 10. The recess 10 shown in Figure 3 is an example of the detailed structure of each of the recesses 10 shown at the top of Figure 1.
[0049] Each recess 10 consists of a bottom portion 11 and an opening portion 12. The bottom portion 11 is a portion that serves as the bottom of the culture chamber 1, and the opening portion 12 is a portion disposed above of the bottom portion 11. A portion in which the bottom portion 11 and the opening portion 12 are in contact is indicated as a boundary. In Figure 2, a portion whose length is indicated by an "R" arrow corresponds to the limit position. In Figure 3, the position of the limit is indicated by a double dashed chained line. It should be noted that the bottom portion 11 and the opening portion 12 are formed with a continuous surface and produced in an integrated manner.
[0050] Figures 2 and 3 show an equivalent diameter R and a depth (height) H of each recess of the plurality of recesses 10 formed in the culture chamber 1.
[0051] The term "equivalent diameter R" refers to the diameter of a circle inscribed on the bottom portion 11 of each recess 10. In this case, the equivalent diameter R is the diameter of an inscribed circle that is inscribed on the boundary between the bottom portion 11 and opening portion 12. More specifically, the equivalent diameter R is the diameter of a circle inscribed in a plane shape that is perpendicular to the direction of height H of each recess 10 at the limit.
[0052] The term "depth D" refers to a length of the bottom within the bottom portion 11 to an upper end of each recess 10. The upper end of the recess 10 corresponds to an end (upper end) of the portion of opening 12. Depth D corresponds to the depth of a space formed by the recess 10. In other words, depth D is a depth from the bottom of a space, which is formed by the bottom portion 11, to an upper end of a formed space by the opening portion 12. Figure 2 shows not only the depth D of the recess 10, but also a depth D1 of the bottom portion 11 and a depth D2 of the opening portion 12.
[0053] The bottom portion 11 forms a space (first space) in which the cells are grown. The bottom portion 11 is, for example, hemispherical in shape. For example, a shape obtained by dividing a spherical shape that has the equivalent diameter R as a diameter in halves can be used. The shape of the bottom portion 11 is not limited to a hemispherical shape. Other specific examples of the format will be described in a second modality.
[0054] Opening portion 12 forms a space (second space) that operates to support culture and cell collection. The opening portion 12 is formed by a wall that surrounds an area of a boundary between the opening portion 12 and the bottom portion 11 to an end (tip) of the recess 10 and which has a tapering angle in a strip of 1 degree to 20 degrees. The tapering angle of the wall constituting the opening portion 12 is preferably in a range of 5 degrees to 15 degrees, and more preferably 10 degrees. This is because, if the tapering angle is extremely small, it is difficult to transfer cells from the recesses to a medium during the collection of the cells and, if the tapering angle is extremely large, the cells are removed during the replacement of the medium.
[0055] The taper angles are represented by 01 and 02 in Figure 2. In an example of the shape of each recess 10 shown in Figures 2 and 3, the taper angles 01 and 02 are substantially the same.
[0056] The boundary between the bottom portion 11 and the opening portion 12 is formed in such a way that the equivalent diameter R is in a range of 50 μm to 1 mm. To supply nutrients to a central portion of a spheroid, the equivalent diameter is preferably in a range of 50 μm to 500 μm and more preferably in a range of 100 μm to 500 μm. This is because it is claimed that nutrients and oxygen are transferred to cells only by diffusion and a central portion of a spheroid with a size of 300 μm or less does not become necrotic (Efrem Curcio et al., "Mass transfer and meta - bolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system ", Biomaterials 28 (2007) 5487-5497). Therefore, the aforementioned diameter range is preferable to prevent a spheroid from reaching the size of 300 μm.
[0057] On the other hand, when it is intended to cause necrosis in a central portion of a cell, such as a cancer cell (Franziska Hirschhaeuser et al., "Multicellular tumor spheroids: An underestimated tool is catching up again", Journal of Biotechnology 148 (2010) 3-15, Figure 1), the equivalent diameter R is preferably equal to or greater than 400 μm and less than 2 mm. This is because, as mentioned above, nutrients can be transferred to a central 300 µm spheroid size, so that necrosis does not occur. Therefore, in order to obtain a spheroid that has a diameter of 300 μm or more, it is necessary that the equivalent diameter is equal to or greater than 400 μm.
[0058] In addition, the depth D from the bottom of the bottom portion to the end of each of the recesses is adjusted in a range 0.6 or more times to 3 or less times the equivalent diameter R. The depth D is preferably in a range of 0.7 or more times to 1.2 or less times the equivalent diameter R and more preferably in a range of 0.8 to 1 times the equivalent diameter R.
[0059] In each culture chamber 1, the area between two adjacent recesses 10 is preferably flat. For example, the distance between two recesses 10 is preferably in the range of 5 μm to 50 μm. This is because it is preferable to increase the number of spheroids per unit area and to cultivate the spheroids to a high density so that a large number of spheroids can be efficiently obtained. To achieve this, the area of the upper wall surface in which no spheroid is formed is preferably as small as possible. In this case, however, when the tapering angle is small and the wall is thin, cracks can easily occur due to vibration during cell seeding or medium replacement. Therefore, the distance between two recesses is preferably 5 μm or more. In view of this, the distance between two recesses is preferably in a range of 5 to 20 μm.
[0060] On the other hand, the two adjacent recesses 10 can come into contact with each other. For example, a part of one end of one of the two recesses 10 and a part of an end of the other of the two recesses 10 can come into contact with each other, so that the inclined surfaces of the opening portions 12, each of which form a tapering angle, can come in contact with each other to form a zigzag shape.
[0061] The culture chamber 1, which has the format described above, is preferably produced as follows.
[0062] Each culture chamber 1 is preferably a resin mold formed from one or a combination of two or more selected from the group consisting of acrylic resin, polylactic acid, polyglycolic acid, styrene resin, acrylic resin of styrene copolymer, polycarbonate resin, polyester resin, polyvinyl alcohol resin, ethylene copolymer resin and vinyl alcohol, thermoplastic elastomer, vinyl chloride resin and silicon resin.
[0063] A functional group is preferably formed in the recesses 10 of the culture chamber 1 by a method of treating surface modification of any of the plasma treatment, the coating with glass, the luminous electrical discharge and the ozonation with UV , or a combination thereof, and the treatment is preferably carried out so that the water contact angle is 45 degrees or less.
[0064] Furthermore, a hydrophilic polymer chain that inhibits cell adhesion is preferably immobilized in recesses 10. More preferably, the hydrophilic polymer chain is immobilized in recesses 10 which are treated so that the contact angle of the above mentioned water is 45 degrees or less.
[0065] Furthermore, a phospholipid or an epolymer phospholipid complex is preferably immobilized in recesses 10. More preferably, this immobilization treatment is carried out on each recess 10 which is treated so that the above-mentioned water contact angle either 45 degrees or less, each recess 10 in which a hydrophilic polymer chain is immobilized, or a combination of these recesses 10.
[0066] In addition, each of the recesses 10 preferably has a cell non-stick surface where at least one polymer of a hydrophilic polymer chain that inhibits cell adhesion, and a phospholipid, or phospholipid complex and polymer is immobilized after a functional group is formed in the recesses by a method of treating surface modification between any of the plasma treatment, the glass coating, the light electric discharge and the UV ozonation, or a combination thereof , and the treatment is carried out so that the water contact angle is 45 degrees or less. Most preferably, this treatment is carried out in conjunction with a treatment or a combination of the treatments mentioned above.
[0067] The hydrophilic polymer chain mentioned above is preferably poly (hydroxyethyl) methacrylate. Most preferably, the average molecular weight of poly (hydroxyethyl) methacrylate is 100,000 or more. <Culture Method>
[0068] Next, a method of culturing cells will be described when using the culture chambers 1 shown in Figures 1 to 3.
[0069] Culturing the cells is carried out by the following steps: a) adding a medium in which the cells are dispersed in the culture chambers 1; b) cell cultivation; c) substitution of the medium; d) placement of spheroids to grow; e) suspension of spheroids in the medium; and f) cell recovery.
[0070] The steps mentioned above can be classified into two steps, that is, the cell culture step (cell culture step) and the cell recovery step (cell collection step). The cell culture step includes steps a) to d), and the cell collection step includes steps e) and f).
[0071] The term "spheroid" used in this document refers to a three-dimensional cluster of cells that includes an aggregated cell number. Each of the steps will be described below. a) Step of adding a medium in which the cells are dispersed in the culture chambers 1
[0072] This step is a preparation step for cell culture. In this step, the total number of cells as described below is dispersed in a medium and added to each culture chamber 1.
[0073] A lower limit of the total number of cells is equal to or greater than the number (n) of the recesses 10 present in the culture chamber 1.
[0074] An upper limit on the total number of cells is equal to or less than a number obtained by multiplying the number (n) of the recesses by a value obtained by dividing the volume (V) of each of the recesses 10 of the culture chamber 1 by the volume (v) of the cells to be seeded. The upper limit of the total number of cells can be represented by the following formula when using the symbols: V / v x n. This is based on the premise that the volumes (V) of the plurality of recesses 10 are the same. If the volumes (V) of the plurality of recesses 10 are different, an average value is used.
[0075] The medium is adjusted depending on the cells to be grown. b) Cell cultivation stage
[0076] The cells are cultured for 12 hours or more in each culture chamber 1 to thereby allow the cells to form a spheroid. When the medium is added to each culture chamber 1, the cells dispersed in the medium are loaded in the recesses 10 and the cells are grown in the respective recesses 10. It is preferable to load one cell in each recess 10, and it is preferable to form a spheroid in space formed by the bottom portion 11. In each recess 10, a cell proliferates in the bottom portion 11 of the recess 10. If at least one cell is not present in each recess during cultivation and seeding, no spheroid is formed in the recess 10, because no cell moves from the adjacent recess 10 to said recess 10 during culture. In order to grow spheroids at a high density, it is preferable to form a spheroid in each recess 10. Therefore, it is preferable to form at least one cell in each recess 10. In terms of production efficiency, it is preferable to reduce the initial number of the cells as much as possible and recover as many spheroids as possible and, therefore, it is preferable that the number of cells present in each recess 10 is as small as possible. For this reason, it is preferable that one cell is present in each recess 10. c) Medium replacement step
[0077] During medium replacement, 20% or more of the medium in each culture chamber 1 is sucked, and the same amount of fresh medium is then injected into the culture chamber. It is preferable to replace the medium at least once during cell culture. d) Stage of placing the spheroids to grow
[0078] Steps a) to c) described above are performed a plurality of times to thereby allow the spheroids to grow. In the case of spheroid differentiation and induction, it is preferable that each spheroid can grow to a size limited by the space formed by the bottom portion 11 of each recess 10 and that the medium is then replaced by the differentiation induction medium to differentiate each way spheroid. In addition, it is more preferable that 60% or more of the total number of spheroids formed in the culture chambers 1 have a diameter in the range of ± 5% of an average spheroid diameter. e) Stage of suspension of spheroids in the middle
[0079] After each spheroid is grown to a desired size, the cells cultured in each recess 11 are suspended in the medium by shaking the medium in each culture chamber 1. For example, this step is performed by shaking the medium. Specifically, shaking the medium can be done by any of the following means: (1) shaking the medium by shaking each culture chamber 1; (2) agitation of the medium when sucking and discharging the medium (pipetting); (3) stirring the medium by arranging a stirring paddle in each culture chamber 1; (4) shaking the medium by placing a shaker in each culture chamber 1; and (5) agitating the medium by a combination of two or more of the means (1) to (4) mentioned above. ) Cell recovery step
[0080] The medium that includes the cells in each culture chamber 1 is sucked by a suction machine, to thereby recover the cells (spheroids) suspended in the medium.
[0081] As described above in the first modality, the cell sowing, the replacement of a medium and the collection of the cells can be performed in the same chamber and, in addition, the spheroids can be recovered from each culture chamber.
[0082] Culturing the cells by using the culture chambers 1 of the first embodiment allows the formation of a spheroid that has a desired size in the bottom portion 11. In addition, the cultured spheroids can be efficiently recovered. Specifically, the structure of each recess 10 including the bottom portion 11 and the opening portion 12 makes it possible to easily maintain a state in which cells that adhere to the bottom portion 11 or are suspended in the middle are prevented from being removed when the medium is sucked during medium replacement. In this way, the removal of cells from the bottom portion 11 can be expected to be suppressed. On the other hand, when collecting cells, when the medium in the bottom portion 11 is sucked and discharged, it can be expected that the medium can flow easily through the opening portion 12. One can also expect the use of the format hemispherical bottom portion 11 contributes to the formation of spheroids with a uniform shape and size. Second Mode
[0083] Although an example of the structure in which the funtion portion of 11 has a hemispherical shape has been described in the first embodiment, other shapes of the bottom portion will be described in a second embodiment. The bottom portion can have any shape, such as a shape formed when using a part of a spherical shape, a truncated cone shape or a linear shape. The linear shape of the bottom portion is a shape that has no substantial bottom portion and that has a recess formed only by an opening portion. Figures 4 to 7 show examples of the shape of each recess according to this modality. Figures 4 to 7 show the recesses 20A to 20D which have the lower portions 21A to 21D, respectively, which are different from the bottom portion 11 of the first embodiment. Since the opening portion 12 can be formed with the same shape as that of the first embodiment, Figures 4 to 7 show examples of the shape of each recess in which the portions of the opening having the same shape are combined.
[0084] Although in the first embodiment a hemispherical shape, which is obtained by dividing a spherical shape into halves, is used for the bottom portion 11, Figures 4 and 5 show examples in which different hemispherical shapes are used for the portion background. Figure 4 shows the bottom portion 21A for which a portion less than half a spherical shape is used. In other words, Figure 4 shows a case where a portion of a hemispherical shape is used for the bottom portion 21A. Figure 5 shows the bottom portion 21B that is cylindrical in shape with a hemispherical bottom. In the case of the shape of the bottom portion 21B shown in Figure 5, as the length of the cylindrical portion increases, cells are less likely to be suspended in the middle of the bottom portion 21B during collection of the cells. Therefore, it is preferable to adjust the length of the cylindrical portion. For example, it is preferable to form the bottom portion 21B and the opening portion 12 so as to maintain the same ratio (1: 1) between the depth (height) of the bottom portion 21B and the depth (height) of the opening portion 12.
[0085] Figure 6 shows the bottom portion 21C for which a truncated cone shape is used. When the bottom portion is flat, the reflection and interference of light can be reduced and is thus useful for observation with a microscope.
[0086] Figure 7 shows an example of the shape of the recess 20D in which the bottom portion 21D has a linear shape, that is, the bottom portion 21D does not form a space. The efficiency of culture and collection of cells in the 20D recess is less than that of culture chambers that have other shapes. However, the recess 20D has an advantage that it facilitates the production process for the culture chambers.
[0087] A case in which the opening portion 12 is formed with a shape similar to that of the first embodiment has been described in this embodiment. However, the present invention is not limited to this case.
[0088] The cell culture method when using the culture chambers according to this modality is similar to that of the first modality, and the description of the same is thus omitted.
[0089] The culture chambers according to this modality can provide the same advantageous effects as those of the first modality. Third Mode
[0090] A mode in which the shape of the opening portion 12 is a circular shape or a substantially circular shape has been described in the above embodiments. A culture chamber that includes opening portions in which each has a shape other than a circular shape or a substantially circular shape will be described. One end of each opening portion may be of a shape other than a circular shape or a substantially circular shape, such as a hemispherical shape, a trapezoidal shape or an inverted triangular shape. On the other hand, it is necessary that the shape of the boundary where the opening portion comes into contact with the bottom portion (the boundary portion of the opening portion) is the same as the shape of the boundary portion of the bottom portion. Figures 8 and 9 show recesses 30A and 30B, respectively, each of which has an end with a different shape than that of the opening portion 12 of the first embodiment. Although Figures 8 and 9 show the same bottom portion 11 as that of the first embodiment, a combination of any of the bottom portions 21A to 21D of the second embodiment, or a bottom portion that has a different shape, can be used. The bottom portion and the opening portion can be of any shape, as long as an inclined surface can be formed continuously at the boundary between the bottom portion and the opening portion.
[0091] Figure 8 shows an example of the shape of an end of the opening portion 32A which is formed in a curve. Figure 8 is a top view of the recess 30A. One end of the bottom portion 11 is indicated by a circle having the equivalent diameter R, and the outer periphery of the opening portion 32A is indicated by a curve. The end of the opening portion 32A has a curved shape that is not symmetrical in the horizontal direction and in the vertical direction. However, the end of the opening portion 32A can be shaped that is symmetrical in the horizontal direction or in the vertical direction. Figure 9 shows an example in which an end of the opening portion 32B is rectangular in shape. Although Figure 9 shows an example where one end of the opening portion is square in shape, the end of the opening portion can be of another polygonal shape, or a combination of a curve and a straight line. Figure 9 is a top view of the recess 30B. The end of the bottom portion 11 is indicated by a circle having the equivalent diameter R, and the outer periphery of the opening portion 32B is indicated by a solid square. For example, the shape of the end of the bottom portion can be modified to fit the area of the space between the end of the bottom portion and the adjacent recess. Since it is necessary for the shape of the end of the opening portion to play a role in promoting cell suspension, the taper angle is important.
[0092] In the shape examples shown in Figures 8 and 9, the taper angle has a value that varies depending on the shape of the opening portions 32A and 32B. This is because the inclination of the inclined surface that forms the wall varies depending on the shape of the opening portions 32A and 32B.
[0093] Each of the shapes of the opening portions illustrated in this embodiment can be combined with the shape of the bottom portion 11 described in the first embodiment, or the shape of the bottom portion described in the second embodiment. In addition, these formats can also be combined with a format other than the background portion formats illustrated in the above modalities, as a matter of expectation.
[0094] The cell culture method that uses the culture chambers according to this modality is similar to that of the first modality, and the description of it is thus omitted.
[0095] Culture chambers according to this modality can provide the same advantageous effects as those of the first modality. Fourth Mode
[0096] Figure 1 illustrates a way in which the culture chambers 1 according to one embodiment are arranged on the culture plate 3 (cavity plate). Culture chambers 1 according to one embodiment can also be formed in a chamber (instrument) other than the culture plate 3 shown in Figure 1. Figures 10 to 12 show examples of the structure of a culture chamber according to a fourth modality. Figure 10 is a schematic view showing an example of the structure in which a flask-shaped culture flask is used. Figure 11 is a schematic view showing an example of the structure in which a culture plate frame is used. Figure 12 is a schematic view showing an example of the structure in which the culture plate shown in Figure 11 is designed in a stack format and used.
[0097] In Figure 10, a bottom surface of a culture flask 4 is used as a culture surface 4A (bottom culture surface). The culture surface 4A corresponds to each culture chamber 1 shown in Figure 1. Therefore, the culture surface 4A can also be indicated as a culture chamber. Like each culture chamber 1 shown in Figure 1, the culture surface 4A is a unit for using the same medium. The culture flask 4 includes a cap 4B. The culture surface area 4A can be designed depending on the intended use. Examples of the size of a typical culture flask include 25, 75, and 225 cm2. A plurality of recesses 40 is formed on the culture surface 4A of the culture flask 4. For example, on the bottom surface of the culture flask 4, a protected area is projected as culture surface 4A and the plurality of recesses 40 is formed on the surface. of culture 4A. The shape of each recess 40 (the shape of each between the bottom portion and the opening portion) can be any of the shapes illustrated in the above embodiments.
[0098] Figure 11 shows an example where only the culture plate frame is used. In Figure 1, the culture chambers 1 (cavities) are formed in the culture plate 3, whereas in Figure 11 a lower surface of a culture plate 5 is used as a culture surface 5A (lower culture surface). The culture surface 5A corresponds to each culture chamber 1 shown in Figure 1. Therefore, the culture surface 5A can also be indicated as a culture chamber. The culture surface 5A is a unit for using the same medium. The bottom part of Figure 11 shows an example (seen in schematic cross section) of the structure of the culture surface 5A. For example, on the bottom surface of the culture plate 5, a protected area is designed as a culture surface 5A and a plurality of recesses 50 are formed on the culture surface 5A. The recesses 50 shown in Figure 11 are illustrated schematically. The number, size, and so on, of the recesses 50 are designed depending on the intended use. The shape of each recess 50 (the shape of each between the bottom portion and the opening portion) can be any of the shapes illustrated in the above embodiments.
[0099] Figure 12 shows a structural example of a cell stack format in which a plurality of culture plates 5 shown in Figure 11 are stacked. In other words, Figure 12 shows an example of a multistage structure. In a case where the cells are grown in a closed system with a larger area, the cell stack format is generally used. Although Figure 12 illustrates an example where the culture plates 5 shown in Figure 11 are stacked, the culture plates 3 shown in Figure 1 can be stacked. In Figure 12, the illustration of a chamber that accommodates the plurality of stacked culture chambers and provides a mechanism for replacing a medium is omitted. For example, a culture chamber that has a typical cell shape can be used as a chamber that accommodates the plurality of culture plates. The explanation of this is omitted in this document. Other modalities
[00100] In the above modalities, the limit between the bottom portion and the opening portion is defined as being parallel to the bottom of each culture chamber. However, it is not necessary for the limit to be parallel to the bottom. For example, the boundary can be sloped with respect to the bottom, or it can be formed on a curve. It is only necessary that sufficient space to form a spheroid can be formed in the bottom portion 11. [Example]
[00101] As for a culture chamber for the cultivation of an aggregate of cells and a method of collecting them, the experiments were carried out according to the following example and comparative example. (1) Chamber of culture
[00102] The culture chambers shown in Table 1 were used. Table 1

[00103] Like the culture chamber of the example, a culture plate was prepared in which cavities (culture chambers 1), each including the recesses 10 shown in Figures 1 to 3, are formed.
[00104] In Table 1, the microchambers correspond respectively to the recesses 10 shown in Figures 1 to 3 and each of the microspaces is a space formed by each recess 10 (microspace). It can be stated that the number of microspaces per well is the number of recesses per well. (2) Culture method
[00105] To calculate a survival rate and a collection rate, which are described later, by image analysis, endodermal cells labeled with GFP fluorescence were used. Endodermal cells, vascular endothelial cells and human mesenchymal stem cells were mixed at a ratio of 10: 5-10: 2, and the cells were cultured for 30 days in an endothelial cell medium kit: EGM-2 BulletKit ( product code CC- 3162: Lonza). The medium was replaced once every two days. (3) Measuring the spheroid survival rate
[00106] All wells were observed with a confocal laser microscope, and the spheroids were recognized by image analysis software. Then, the number of recognized spheroids was counted and the number was determined as the number of spheroids. The spheroid survival rate was calculated using the following expression.
[00107] Spheroid survival rate (%) = (number of spheres) x 100 / (number of microspaces)
[00108] A few hours after the cells were seeded (day 0), a spheroid-like cluster was formed in 90% or more of the microspaces in all culture chambers. A value obtained by dividing the number of spheroids obtained after replacing the medium on the 10th and 20th days of culture by the number of spheroids obtained on day 0 was determined as the spheroid survival rate. (4) Collection method
[00109] After completing the culture, the solution was stirred with a pipette (manufacturer, model number), and the suspended spheroids were recovered. For example, a pipette capable of sucking at most 1 ml of the medium is used appropriately for a 24-well plate containing 500 μl to 1 ml of the medium. (5) Collection efficiency
[00110] Before and after the collection of the spheroids, images of the spheroids were taken by a confocal laser microscope. (6) Results.
[00111] Figure 13 shows the spheroid survival rate during medium replacement. The vertical axis represents the spheroid survival rate (sphere number) and the horizontal axis represents the number of days of culture.
[00112] Figure 13 shows the data obtained from the beginning of the culture until the 20th day of culture. As shown in Figure 13, the spheroid survival rate in the comparative example decreased significantly compared to that in the example. After culture for 20 days, the survival rate of the spheroids in the example was 60% or more. This indicates that the spheroid survival rate in the example was improved 1.5 times over that in the comparative example.
[00113] Figure 14 shows the images of the spheroids obtained before and after replacing the medium in the example and in the comparative example. Figure 14 shows the images of the spheroids in the culture chamber before and after the second medium replacement on the fourth day of culture. The left side of Figure 14 shows a photograph of the example (p-HEMA from Kuraray), and the right side of Figure 14 shows a photograph of the comparative example (Iwaki MPC). The upper part of the Figure shows the images taken before replacing the medium, and the lower part (below an arrow) of Figure 14 shows the images taken after replacing the medium. More specifically, the images at the bottom of Figure 14 show the state after the replacement of the medium has been performed twice, that is, half of the medium has been replaced (replacement of the middle half), from the state before the replacement of the medium.
[00114] In the images, the white dots correspond to the spheroids. Before replacing the medium, spheroids are confirmed throughout the area. After replacing the medium, in the example, there is no big difference in the number of spheres until and after replacing the medium, and almost all spheroids survived, whereas in the comparative example only about half of the spheroids survived.
[00115] Figure 15 shows the images taken before and after collecting the cells in the example. The left side (BEFORE) in Figure 15 shows the image of the cells taken before collecting the cells, and the right side (AFTER) in Figure 15 shows the image of the cells taken after collecting the cells. The upper part of Figure 15 shows the image of the entire culture chamber, and the lower part of Figure 15 shows the enlarged image of a part of the culture chamber. Figure 16 shows photographs of the spheroids recovered from the culture chamber according to the example.
[00116] The dot-like portions in each black circular microchamber (recess) correspond to the spheroids. The non-dot-like portions are found in the image taken after collecting the cells, which indicates that almost 100% of the cells can be recovered. In addition, as shown in Figure 16, the recovered cells 20 have an excellent spheroid shape, and each spheroid was not destroyed by the collection operation.
[00117] It should be noted that the present invention is not limited to the modalities described above. Those skilled in the art can easily make modifications, additions and conversions to each component in the above modalities within the scope of the present invention.
[00118] The present patent application is based on and claims the benefit of the priority of Japanese Patent Application no. 2013 120915, filed on June 7, 2013, the disclosure of which is incorporated into this document as a reference in its entirety. List of Reference Signs 1 1 CULTURE CHAMBER 3, 5 CULTURE PLATE 4A, 5A CULTURE SURFACE 5 CULTURE BOTTLE 10, 20A to 20D, 30A, 30B, 40, 50 UNDER 11, 21A to 21D BACKGROUND PORTION 12, 32A, 32B OPENING PORTION

权利要求:
Claims (14)
[0001]
1. Culture chamber, which comprises: a cavity or flask, the cavity or flask comprising a plurality of recesses (10) formed at the bottom of the cavity or flask, each recess having a bottom portion (11) and an opening portion (12), characterized by the fact that the bottom portion (11) of the recess comprises a hemispherical shape, a cylindrical shape with a hemispherical bottom or a truncated cone shape, each of the plurality of recesses (10) comprises a non -adhesive of the cell in which a poly (hydroxyethyl methacrylate) is immobilized after a functional group is formed in the recesses (10) by a method of treating surface modification of any of the plasma treatment, the coating with glass, luminous electrical discharge and ozonation with UV, or a combination thereof, and the treatment is carried out so that a water contact angle is 45 degrees or less, the opening portion (12) of the recess is defined through a wall and which surrounds an area of a boundary between the opening portion and the bottom portion of the recess to an end of each recess, the wall comprising a tapering angle in a range of 10 degrees to 20 degrees, an equivalent diameter of The limit is in a range of 50 μm to 2 mm and a depth from the bottom of the bottom portion to the end of each of the recesses is in the range of 0.6 or more times to 3 or less times the equivalent diameter, and the wall defining the opening portion (12) of the recess forms a continuous surface to the bottom portion, and an inclination of the continuous surface changes in the tapering angle of the wall of the opening portion is greater than a tapering angle of the door. bottom edge, and an area between two adjacent recesses is flat and a distance between the two recesses is in the range of 5 μm to 50 μm.
[0002]
2. Culture chamber, according to claim 1, characterized by the fact that the end of each recess comprises one selected from the group consisting of a hemispherical shape, a trapezoidal shape and an inverted triangular shape.
[0003]
3. Culture chamber, according to any one of the vindications 1 to 2, characterized by the fact that the culture chamber is a resin mold formed from one or a combination of two or more selected from the group consisting of a acrylic resin, a polylactic acid, a polyglycolic acid, a styrene resin, a styrene copolymer acrylic resin, a polycarbonate resin, a polyester resin, a polyvinyl alcohol resin, an ethylene copolymer resin and vinyl alcohol, a thermoplastic elastomer, a vinyl chloride resin and a silicon resin.
[0004]
4. Culture chamber, according to claim 1, characterized by the fact that a phospholipid or a phospholipid-polymer complex is immobilized in the recesses.
[0005]
5. Culture chamber according to claim 9, characterized by the fact that the average molecular weight of poly (hydroxyethyl) methacrylate is 100,000 or more.
[0006]
6. Method for sowing cells, culturing cells, replacing a medium and collecting cells characterized by the fact that it comprises: dispersing cells in a medium, where a total number of cells is equal to or greater than an N number of the recesses of the culture chamber as defined in claim 1 and equal to or less than a number obtained by multiplying the number N of the recesses by a value obtained by dividing a volume V1 of a space defined by each of the recesses by a volume V2 of cells to be seeded; and add the medium to the culture chamber.
[0007]
7. Culture method, according to claim 6, characterized by the fact that a spheroid is formed in a space defined by each of the recesses.
[0008]
8. Culture method, according to claim 7, characterized by the fact that a spheroid is placed to grow.
[0009]
9. Culture method according to claim 6 or 7, characterized by the fact that a spheroid is differentiated and induced.
[0010]
10. Culture method according to any one of claims 6 to 9, characterized in that 60% or more of a total number of spheroids formed in the culture chamber have a diameter in a range of ± 5% of a diameter of medium spheroid.
[0011]
11. Culture method according to any of claims 6 to 9, characterized by the fact that cells in the recesses are recovered by shaking the medium.
[0012]
12. Culture method according to claim 11, characterized by the fact that the medium is agitated by any of the following media selected from the group consisting of agitation of the medium when agitating the culture chamber; agitation of the medium when sucking and discharging the medium; stirring the medium by arranging a stirring paddle in the culture chamber; and shaking the medium by placing a stirrer in the culture chamber, or a combination thereof.
[0013]
13. Culture method according to any one of claims 6 to 12, characterized in that the medium is replaced at least once, and 20% or more of the medium is replaced.
[0014]
14. Culture method for sowing cells, culturing cells, replacing a medium and collecting cells, characterized by the fact that it comprises: dispersing cells in a medium, where the number of cells is equal to or greater than one number n of the recesses of the culture chamber as defined in claim 1 and equal to or less than a number obtained by multiplying the number n of the recesses by a value obtained by dividing a volume V of each of the recesses by a volume v of the cells a be sown, and addition of the medium to the culture chamber; culturing the cells in the culture chamber for 12 hours or more to form a spheroid; suck 20% or more of the medium and then inject the same amount of fresh medium; repeat dispersion, cultivation and suction a plurality of times to allow the spheroid to grow; allowing the spheroid to grow to a desired size and then shaking the medium to suspend the cells within the recesses in the medium; and sucking the medium comprising the cells by a suction machine to recover the cells.

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AU2014276229B2|2019-11-21|

BR112015030041A2|2017-07-25|

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AU2020201221A1|2020-03-12|

AU2020201221B2|2021-12-23|

CN105308170A|2016-02-03|

WO2014196204A1|2014-12-11|

US20200063080A1|2020-02-27|

CA2914463A1|2014-12-11|

KR20160017036A|2016-02-15|

EP3006553A4|2017-01-04|

KR102359408B1|2022-02-07|

AU2014276229A1|2016-01-21|

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法律状态:
2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

2019-09-17| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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JP2013120915|2013-06-07|

JP2013-120915|2013-06-07|

PCT/JP2014/002993|WO2014196204A1|2013-06-07|2014-06-05|Culture vessel and culture method|

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