WO2017209301A1 - Well plate, well plate sheet, and culturing method - Google Patents

Well plate, well plate sheet, and culturing method Download PDF

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Publication number
WO2017209301A1
WO2017209301A1 PCT/JP2017/020715 JP2017020715W WO2017209301A1 WO 2017209301 A1 WO2017209301 A1 WO 2017209301A1 JP 2017020715 W JP2017020715 W JP 2017020715W WO 2017209301 A1 WO2017209301 A1 WO 2017209301A1
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Prior art keywords
pattern
well plate
well
culture
concave
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PCT/JP2017/020715
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French (fr)
Japanese (ja)
Inventor
優一 矢代
良樹 越仮
渡沼　宏至
吉隆 高濱
谷　敍孝
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Jsr株式会社
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Publication of WO2017209301A1 publication Critical patent/WO2017209301A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

Definitions

  • the present invention relates to a well plate, a well plate sheet and a culture method.
  • culturing cells on a plate has been widely performed for the purpose of production of compounds and use in the medical field.
  • improvements are being made on the three-dimensional cell culture technique that can obtain a cell mass closer to the state in the living body.
  • a three-dimensional cell culture technique include a technique of culturing a spherical cell mass in each well using a well plate having a plurality of wells (see JP 2013-215152 A).
  • a porous membrane coated with collagen having the property of easily adhering to cells and promoting cell growth is used. Since the cells attached to the surface of the collagen laminated porous membrane grow not only in the normal plane direction but also in the height direction, this collagen laminated porous membrane is arranged at the bottom of the well.
  • the cell mass can be grown spherically in the well.
  • the present invention has been made on the basis of the circumstances as described above, and an object of the present invention is to provide a well plate that can be easily manufactured and that can easily obtain a minute cell mass with high size uniformity, and the well. It is an object of the present invention to provide a well plate sheet used for producing a plate and a culture method using the well plate.
  • the invention made to solve the above-mentioned problems is a well plate having one or a plurality of wells for containing and culturing cells, wherein the wells have a concavo-convex pattern on the bottom surface, and the concavo-convex pattern is a concavo-convex pattern. It is a composite pattern of a first pattern constituted by a combination and a second pattern constituted by a combination of unevenness finer than the first pattern, and the second pattern is formed on the surface of the first pattern. It is characterized by being.
  • a sheet that forms the well bottom surface of a well plate including one or a plurality of wells that contain and culture cells, and includes a base film,
  • the base film has a concavo-convex pattern in at least the well region of one surface, and the concavo-convex pattern is a combination of a concavo-convex finer than the first pattern, and a first concavo-convex pattern formed by a combination of concavo-convex patterns It is a composite pattern with the 2nd pattern comprised, The said 2nd pattern is formed in the surface of the said 1st pattern, It is characterized by the above-mentioned.
  • Yet another aspect of the present invention made to solve the above-mentioned problems is a method for containing and culturing cells, wherein the well plate is used, and the well plate is subjected to the average diameter of the cell mass obtained by the culture.
  • the culture method is characterized in that the flat filling factor of the first pattern is adjusted.
  • the well plate is easy to manufacture, and according to the well plate, a cell mass with high size uniformity can be easily obtained. Further, according to the well plate sheet, the well plate can be easily manufactured. Furthermore, since the culturing method uses the well plate, it is possible to easily cultivate a cell mass having high size uniformity.
  • the well plate can be easily fixed to the well plate by the cell mass generated by the culture by using the second pattern having fine irregularities.
  • FIG. 2 is a schematic enlarged partial sectional view taken along line AA of the well plate of FIG. 1.
  • 2 is a laser micrograph showing the well bottom of the well plate of Example 1.
  • FIG. 2 is a laser micrograph showing a fine uneven pattern of a well plate of Example 1.
  • the well plate includes a plurality of wells 1 for containing and culturing cells.
  • the well 1 includes a bottom surface 2 and an inner peripheral surface 3, and the bottom surface 2 of each well 1 has an uneven pattern.
  • the concavo-convex pattern includes a first pattern 1a configured by a combination of concavo-convex and a second pattern (fine concavo-convex pattern) that is configured by a combination of concavo-convex finer than the first pattern 1a and combined with the first pattern 1a. 1b.
  • the second pattern 1b is formed on the surface of the first pattern 1a
  • the second pattern 1b is formed on the surface of the first pattern 1a.
  • “Fine unevenness” means that the average pitch and average height difference (average horizontal distance and average vertical distance between the lowest point of the adjacent valley and the highest point of the peak) are small.
  • the well plate is stacked between the well plate body 4 constituting the side wall of the well 1, the bottom plate 5 constituting the bottom wall of the well 1, and the well plate body 4 and the bottom plate 5.
  • a well plate sheet 6 constituting the bottom surface 2 is mainly provided.
  • the well plate body 4 has a thick plate shape having a plurality of through holes 7. Further, the concave and convex concave portions constituting the first pattern 1 a are formed on the surface of the well plate sheet 6.
  • the shape of the well plate in plan view includes a rectangular shape and a circular shape, but a rectangular shape is preferable.
  • the length of the short side and the long side in the plane direction can be adjusted as appropriate according to the size and arrangement density of the wells 1 and the size of the equipment used for culture. For example, for 96, 384 and 1536 wells, there are ANSI / SBS 1-2004 to 4-2004 standards.
  • the main component of the well plate body 4 and the bottom plate 5 is not particularly limited as long as it does not adversely affect the cells to be cultured and does not change in quality by the culture solution.
  • polypropylene, polyester, polyolefin, polystyrene, polyurethane examples thereof include fluorine-containing resins such as cellulose, regenerated cellulose, polycarbonate, polyamide, polyimide and polytetrafluoroethylene.
  • the “main component” means the most abundant component (for example, 50% by mass or more) on a mass basis.
  • the well plate body 4 and the bottom plate 5 may contain various additives in addition to the main components.
  • the well plate body 4 and the bottom plate 5 may be made of the same material or different, but are preferably made of the same material. Since the well plate body 4 and the bottom plate 5 are made of the same material, the well plate body 4 and the bottom plate 5 can be easily joined in the joining step described later.
  • Examples of the shape of the well 1 include a prismatic shape, a truncated pyramid shape, a cylindrical shape, a truncated cone shape, an elliptical columnar shape, and an elliptical truncated cone shape whose axis is a direction perpendicular to the bottom surface of the well plate.
  • the bottom surface of the well plate means “the surface of the well plate body 4 where the well plate body 4 and the well plate sheet 6 are in contact”.
  • the shape of the well 1 is preferably a columnar shape and a truncated cone shape, more preferably a truncated cone shape, and more preferably a truncated cone shape having a small bottom side and a large opening side.
  • the inner diameter of the well 1 gradually increases from the bottom surface 2 toward the opening.
  • the well plate can be easily removed from the mold when the well plate body 4 is injection-molded, and the manufacture of the well plate becomes easier.
  • the opening is relatively large, introduction of the culture medium into the well 1 and various operations during cell culture are facilitated.
  • the lower limit of the bottom surface diameter D1 of each well 1 is preferably 0.5 mm, more preferably 0.8 mm, and even more preferably 1 mm.
  • the upper limit of the bottom diameter D1 is preferably 10 cm, more preferably 5 cm, and even more preferably 3.5 cm. If the diameter D1 is smaller than the lower limit, introduction of the culture medium into the well 1 may be difficult. Conversely, if the bottom diameter D1 exceeds the upper limit, the well 1 becomes excessively large relative to the size of the cell mass that can be three-dimensionally cultured, and as a result, the space that does not contribute to cell culture increases. There is a possibility that the culture efficiency when performing the three-dimensional culture by using it decreases.
  • the bottom diameter D1 of the well 1 when the cross section of the well 1 is an ellipse means the average value of the major axis and the minor axis, and the bottom diameter D1 of the well 1 when the cross section of the well 1 is a rectangle. Mean the average value of the long and short sides.
  • the upper limit of the flatness is preferably 0.2 and more preferably 0.15. If the flatness exceeds the above upper limit, the space that does not contribute to cell culture increases and the culture efficiency may decrease, or the symmetry of the shape of the resulting cell mass decreases, making it difficult to obtain many homogeneous cell masses. There is a risk of becoming.
  • the “flattening ratio” means a value represented by (ab) / a where the major axis radius of the ellipse is a and the minor axis radius is b.
  • the lower limit of the ratio of the long side to the short side is preferably 1.
  • the upper limit of the ratio is preferably 1.3 and more preferably 1.2. If the ratio exceeds the above upper limit, the space that does not contribute to cell culture increases and the culture efficiency may decrease, the symmetry in the shape of the resulting cell mass decreases, and it is difficult to obtain a large number of homogeneous cell masses There is a risk.
  • the lower limit of the depth of each well 1 is preferably 0.5 mm, more preferably 1 mm, and even more preferably 1.2 mm.
  • the upper limit of the depth is preferably 2 cm, more preferably 1.8 cm, and even more preferably 1.5 cm.
  • the depth is smaller than the lower limit, it is difficult to stably hold the culture solution in the well 1 and it may be difficult to perform three-dimensional culture of cells.
  • the depth exceeds the upper limit, it is difficult to take out the cell mass after culturing from the well 1.
  • the lower limit of the volume of each well 1 is preferably 0.1 ⁇ L, more preferably 0.5 ⁇ L, and even more preferably 0.8 ⁇ L.
  • the upper limit of the volume is preferably 10 mL, more preferably 7 mL, and even more preferably 4 mL. If the volume is smaller than the lower limit, introduction of the culture solution into the well 1 may be difficult. On the contrary, when the diameter exceeds the upper limit, the well 1 becomes excessively large relative to the size of the cell mass that can be three-dimensionally cultured, and the amount of the culture medium to be used is unnecessarily increased. The associated costs may increase.
  • the shapes of the plurality of wells 1 are the same. As described above, when the well plate includes a plurality of wells 1 having the same shape, a large number of cell clusters having excellent shape homogeneity can be obtained by a single culture procedure.
  • each well 1 has a plurality of recesses constituting the first pattern 1a, and this recess has a second pattern 1b which is a fine uneven pattern on the entire surface (inner surface).
  • a cell cluster having a size (small CV value) can be obtained.
  • the second pattern 1b formed on the surface of the concave portion allows the seeded cells to grow many provisional limbs, and the adhesion to the well 1 is improved. This cell grows in a planar direction along the surface (inner surface) of the recess, and continues to grow in the height direction using the portion grown in the recess as a scaffold, so that a cell mass is efficiently obtained. be able to.
  • the composite uneven pattern of the first pattern 1a and the second pattern 1b exists as a part of the bottom surface 2 once formed on the bottom surface 2, and hardly deteriorates during storage or cell culture. Therefore, maintenance of the bottom surface 2 of the well is facilitated, and as a result, the cells can be three-dimensionally cultured by a simple procedure.
  • this composite uneven pattern can be formed on the bottom plate 5 and the well plate sheet 6 before being joined to the well plate body 4, and the uneven patterns constituting the first pattern 1a in the fine well 1 can be formed. There is no need to perform the operation of forming the recess. Therefore, the well plate is easy to manufacture.
  • the shape of the concave / convex concave portions constituting the first pattern 1a can be a polygon such as a circle, an ellipse, or a rectangle, but is preferably a circle.
  • the concave portion is circular, the shape of the cultured cell mass can be made closer to a sphere, and as a result, a homogeneous cell mass can be easily obtained.
  • the lower limit of the average diameter D2 of the concave and convex portions constituting one first pattern 1a in each well 1 is preferably 30 ⁇ m, more preferably 100 ⁇ m, and even more preferably 150 ⁇ m.
  • the upper limit of the average diameter D2 is preferably 3000 ⁇ m, more preferably 2000 ⁇ m, and even more preferably 1000 ⁇ m. If the average diameter D2 is smaller than the lower limit, the size of the obtained cell mass may be excessively small. On the other hand, if the average diameter D2 exceeds the upper limit, the space that does not contribute to cell culture during 3D culture increases, and the cell culture efficiency may decrease, or the cells may grow excessively in a plane. May be difficult to culture.
  • the “average diameter of the recesses” means an average value of the diameters of perfect circles having the same area as the planar view area of the recesses.
  • the plane filling rate of the concave and convex recesses constituting the first pattern 1a in each well 1 varies depending on the cell type and the number of seeding when culturing, but the lower limit of the plane filling rate. Is preferably 5%, more preferably 10%.
  • the upper limit of the plane filling rate is preferably 99.9% and more preferably 90%. If the plane filling rate is smaller than the lower limit, the number of cells that do not enter the recess increases as the number of seeding increases, which may increase the CV value (variation) of the cell mass. On the other hand, when the plane filling rate exceeds the upper limit, it is difficult to stably mold the first pattern 1a.
  • the “planar filling ratio of the recesses” means the ratio of the total area in plan view of the recesses in the wells to the area of the bottom surface of one well.
  • the shape of the concave and convex recesses constituting the first pattern 1a is preferably a three-dimensional shape with the direction perpendicular to the bottom surface of the well plate as an axis, from the viewpoints of manufacturability and cell culture.
  • a flat bottomed cylindrical shape such as a truncated pyramid shape, a cylindrical shape, a truncated cone shape, an elliptical columnar shape, an elliptical truncated cone shape, a pyramidal shape, a conical shape, a hemispherical shape, and a semi-elliptical spherical shape are more preferable.
  • a flat bottomed cylindrical shape such as a truncated pyramid shape, a cylindrical shape, a truncated cone shape, an elliptical columnar shape, an elliptical truncated cone shape, a pyramidal shape, a conical shape, a hemispherical shape, and a
  • the lower limit of the maximum depth of the concave and convex portions constituting the first pattern 1a is preferably 20 ⁇ m, and more preferably 50 ⁇ m.
  • the upper limit of the maximum depth is preferably 2000 ⁇ m, and more preferably 1000 ⁇ m. If the maximum depth is smaller than the lower limit, the effect of improving the three-dimensional culture efficiency by the recesses may be insufficient. Conversely, if the maximum depth exceeds the upper limit, it may be difficult to take out the cultured cells from the recess.
  • the lower limit of the radius of curvature is preferably 10 ⁇ m, and more preferably 25 ⁇ m.
  • the upper limit of the radius of curvature is preferably 1000 ⁇ m, and more preferably 500 ⁇ m. If the curvature radius is smaller than the lower limit, it may be difficult to take out the cultured cells from the recess. On the other hand, when the curvature radius exceeds the upper limit, the depth becomes excessively small with respect to the average diameter of the recess, and the effect of improving the three-dimensional culture efficiency by the recess may be insufficient.
  • the concave portions may be regularly arranged. That is, the first pattern 1a is preferably a regular pattern.
  • the plurality of concave portions are regularly arranged, when the plurality of cell masses are cultured, the respective cell masses are arranged at equal intervals. Therefore, an unexpected change in the size of the cell mass due to contact and fusion between adjacent cell masses can be reduced. As a result, the uniformity of the quality of the obtained cell mass can be further improved.
  • these concave portions may have a single shape in plan view.
  • the shape of the cell mass cultured in each said recessed part can be made more uniform. As a result, the uniformity of the quality of the obtained cell mass can be further improved.
  • the cross-sectional shape of the concave and convex portions constituting the plurality of first patterns 1a is a single shape.
  • corrugated recessed part which comprises each 1st pattern 1a can be made more uniform because the cross-sectional shape of several said recessed part is a single shape.
  • the uniformity of the quality of the obtained cell mass can be further improved.
  • the second pattern 1b is composed of a concave region and a convex region that are finer than the concave and convex portions constituting the first pattern 1a, and the width and interval of the concave region and the convex region are each 10 ⁇ m or less, and the depth is 10 nm or more. Pattern.
  • the specific shape of the second pattern 1b is not particularly limited.
  • the planar shape of the concave region and the convex region can be a circle or a polygon, and the lattice shape or honeycomb shape is preferable as the overall planar shape. Can be used for
  • the shape of the concave-convex recesses constituting the second pattern 1b is preferably a hemispherical (lens shape) or flat bottomed cylindrical shape from the viewpoints of manufacturability and cell culture.
  • planar filling rate of the concave and convex portions constituting the second pattern 1b in each well 1 varies depending on the cell type at the time of culturing, but the lower limit is 1% Is preferable, and 20% is more preferable. On the other hand, the upper limit of the plane filling rate is preferably 99%, more preferably 85%. When the plane filling rate is out of the above range, the adhesion of the cell mass is deteriorated and cell culture may be difficult.
  • Plan view area of one of the recesses of the irregularities constituting the second pattern 1b varies depending on the cell types at the time of carrying out the cultivation, as the upper limit, preferably 100 [mu] m 2, 50 [mu] m 2 is more preferable. If the area in plan view is larger than this, the adhesion of the cell mass may be deteriorated. On the other hand, the lower limit of the plan view area, preferably 1 [mu] m 2, 2 [mu] m 2 is more preferable. If the area in plan view is smaller than this, it is difficult to stably form the second pattern 1b.
  • the upper limit preferably 100 [mu] m 2, 50 [mu] m 2 is more preferable. If the area in plan view is larger than this, the adhesion of the cell mass may be deteriorated.
  • the lower limit of the plan view area preferably 1 [mu] m 2, 2 [mu] m 2 is more preferable. If the area in plan view is smaller than this, it is difficult
  • the concave and convex concave portions constituting the plurality of second patterns 1b are regularly arranged. That is, the second pattern 1b is preferably a regular pattern. Moreover, it is good for these recessed parts to have a single shape by planar view. By doing in this way, the uniformity of the quality of the obtained cell mass can further be improved.
  • the method for forming the second pattern 1b is not particularly limited.
  • the second pattern 1b may be formed using sandblasting, etching, ion beam, electric discharge, laser, nanoimprint, embossing, injection molding, screen printing, offset printing, self-organization technology, or the like.
  • nanoimprint technology is used.
  • the concave and convex recesses constituting one first pattern 1a may have a plurality of types of second patterns 1b having different patterns on the surface thereof. Further, the plurality of types of second patterns 1b are preferably regular. Thereby, the adhesiveness to the well 1 of a cell can further be improved.
  • the second pattern 1b may be formed in a portion (flat portion) other than the concave and convex portions constituting the first pattern 1a on the bottom surface 2 of the well 1, but the cells are In order not to protrude from the recess, it is preferably formed only on the bottom surface portion of the recess (not formed on the side surface or outside of the recess of the unevenness constituting the first pattern 1a).
  • the well plate sheet 6 is a sheet that forms a well bottom surface 2 of a well plate that includes a plurality of wells 1 that contain cells and are three-dimensionally cultured, and includes a base film, It has the uneven
  • the well plate sheet 6 is used as the well plate of the bottom plate 5.
  • the well plate main body 4 is laminated on the surface to which the main body 4 is bonded, and then the well plate main body 4 is bonded to the surface of the well plate sheet 6 so that the concave and convex portions constituting the first pattern 1a are exposed in the through holes 7.
  • the well plate having the concave portion on the bottom surface 2 of the well 1 can be easily manufactured.
  • the concave and convex portions constituting the first pattern 1a adjusted to the size of the well 1 are adjusted to the size of the well plate. Since it can be formed on the surface, the well plate can be easily manufactured even when the well 1 is very small.
  • the main component of the well plate sheet 6 is not particularly limited as long as it does not inhibit the growth of cell mass, but the same resin as the main component of the well 1 can be used.
  • polyolefin is preferable, and cyclic olefin polymer is more preferable.
  • Such a cyclic olefin polymer does not inhibit cell growth in cell culture, and can easily form a fine uneven pattern (second pattern).
  • Examples of such a cyclic olefin polymer include ARTON (manufactured by JSR Corporation), ZEONOR (manufactured by Nippon Zeon Corporation), and the like.
  • the manufacturing method of the well plate is not particularly limited.
  • the well plate body 4, the bottom plate 5, and the well plate sheet 6 are separately manufactured and bonded, or the well plate is formed on the well plate sheet 6.
  • Examples include insert molding that directly molds the plate body 4.
  • a method in which the well plate body 4, the bottom plate 5, and the well plate sheet 6 are separately manufactured and bonded is preferable.
  • the well plate can be produced while suppressing damage to the fine uneven pattern on the well plate sheet 6.
  • this method includes a well plate sheet forming step, a well plate body forming step, and a laminating step.
  • the method for forming the well plate sheet 6 is not particularly limited, and examples thereof include a method for forming the concave and convex concave portions and the second pattern 1b constituting the first pattern 1a by the method such as etching exemplified in the description of the second pattern 1b. It is done.
  • a concave / convex concave portion constituting the first pattern 1a is formed on the base film on which the second pattern 1b is formed on one surface by applying a mold having the concave pattern from the opposite surface of the second pattern 1b.
  • the well plate sheet 6 is obtained.
  • the second pattern 1b can be stably formed on the film.
  • an uneven pattern may be formed in a region other than the well region of the well plate sheet 6.
  • the concavo-convex pattern outside the well region is filled by bonding of the well plate body 4 described later.
  • the well plate body 4 having a plurality of through holes 7 is molded with resin.
  • the inner peripheral surface of the through hole 7 corresponds to the inner peripheral surface 3 of the well 1.
  • the resin molding method a known method can be adopted, for example, a method of press molding a resin plate, a method of pouring a liquid resin into a mold and curing it, a method of injection molding of a resin into a mold, etc. Of these, the method of injection molding is preferred.
  • the well plate body 4 having the through holes 7 can be easily molded.
  • the well plate body 4, the well plate sheet 6, and the bottom plate 5 are laminated and joined together.
  • the well plate body 4 is joined to the surface on the well plate sheet 6 side of the laminate of the bottom plate 5 and the well plate sheet 6.
  • a known method can be adopted, for example, adhesion using an adhesive or a tape, pressure bonding by a hot press, ultrasonic wave or laser.
  • adhesion using an adhesive or a tape for example, melt adhesion by the above.
  • pressure bonding by hot press or the like, and melt bonding by ultrasonic waves or lasers are preferable.
  • the culture method is characterized by using the well plate and adjusting the plane filling rate of the recesses of the well plate according to the average diameter of the cell mass obtained by the culture.
  • the cells grow three-dimensionally in the recesses on the bottom surface of the well as described above. Therefore, by adjusting the plane filling rate of the recesses (first pattern) of the well plate according to the average diameter of the cell mass obtained by three-dimensional culture, a cell mass of a desired size can be easily obtained.
  • the variation of the cell mass obtained by the three-dimensional culture can be expressed by a CV value.
  • the CV value is a value (%) obtained by dividing the standard deviation ⁇ of the cell mass size by the average value. It can be said that the smaller the CV value, the higher the uniformity of the cell mass. This CV value varies depending on the filling ratio of the concave portion in plan view.
  • a known one can be adopted. Specific examples include a step of introducing a culture solution into each well of the well plate, a step of dispersing cells to be cultured into each well, and a step of culturing the cells in each well.
  • the well plate, the well plate sheet and the culture method are not limited to the above embodiment.
  • the 1st pattern was set as the form which has a recessed part, it may have a some convex part instead of a recessed part, and may form a 2nd pattern in the surface (outer surface) of this convex part.
  • the 2nd pattern which consists only of a recessed part may be formed in the surface of the said recessed part
  • the 2nd pattern which consists only of a convex part may be formed in the surface of the said convex part.
  • the number of wells provided in the well plate may be one. Further, only one concave / convex concave portion or convex portion constituting the first pattern may be formed per well as in the above-described embodiment, or a plurality may be formed per well. As described above, when one well has a plurality of concave portions or convex portions, the cell mass can be cultured in each concave portion or between the convex portions, so that a fine cell mass can be easily obtained as in the above embodiment. it can.
  • the manufacturing method of the well plate may further include a step of performing a surface treatment on the well plate body or the bottom plate.
  • the surface treatment include a treatment for improving wettability on the surface of the well plate main body or the bottom plate.
  • a treatment for improving wettability on the surface of the well plate main body or the bottom plate include ultraviolet irradiation, a plasma process, etc. are mentioned, for example.
  • the well plate, the well plate sheet and the culture method are not limited to three-dimensional culture, and can be suitably used for two-dimensional culture.
  • Example 1 Using a “ZeonorFilm, ZF14” (manufactured by ZEON Corporation) having an average thickness of 188 ⁇ m as a base film, a fine molding apparatus (FLAN200) made by SCIVAX is used, and a fine uneven pattern (second pattern) is formed on the surface of the inverted shape. Molded using a mold.
  • the shape of the second pattern is a shape in which the recess has a square area in plan view of 9 ⁇ m 2 and each recess is surrounded by a protrusion having a width of 0.7 ⁇ m and a height of 1 ⁇ m.
  • the base film is first heated to 170 ° C., the mold is pressed against the surface of the base film, and is held for 60 seconds when the pressure reaches 4.0 MPa, and then the mold pressure is kept constant. However, it was performed by cooling to 100 ° C. or lower and releasing from the film.
  • the substrate film was heated again to 170 ° C., a hemispherical mold having a diameter of 0.7 mm and a depth of 0.2 mm was pressed against the back surface of the substrate film, and when the pressure reached 4.0 Mpa, 60 seconds. Retained. Thereafter, while keeping the pressure of the mold constant, the mold was cooled to 100 ° C. or less and released from the film to form a recess (first pattern).
  • the flat filling factor of the first pattern in one well was 30%. Further, the planar filling factor of the second pattern in one well was 66%.
  • the obtained sheet for well plate having an average thickness of 188 ⁇ m was fixed in a petri dish having a diameter of 65 mm to prepare a cell culture container.
  • D-MEM medium containing 10% fetal bovine serum, 1.2 g / l sodium bicarbonate, 100 IU / ml penicillin, and 100 ⁇ g / ml streptomycin
  • 4000 cell lines HT-29 cells were seeded. When this was cultured at 37 ° C. in an atmosphere of 5% CO 2 for 5 days, a cell mass was formed. The medium was easily exchanged by simply removing the medium using a micropipette and adding a new medium.
  • Example 2 A cell culture vessel was prepared in the same manner as in Example 1 except that a hemispherical mold having a diameter of 0.3 mm and a depth of 0.05 mm was used instead of the hemispherical mold having a diameter of 0.7 mm and a depth of 0.2 mm. Produced.
  • the flat filling factor of the first pattern in one well was 13%.
  • Example 2 The same procedure as in Example 1 using a plate (“EZ SPHERE dish 35 mm” manufactured by AGC Techno Glass Co., Ltd.) having only the concave and convex portions constituting the first pattern without having the fine concave and convex pattern (second pattern).
  • a plate (“EZ SPHERE dish 35 mm” manufactured by AGC Techno Glass Co., Ltd.) having only the concave and convex portions constituting the first pattern without having the fine concave and convex pattern (second pattern).
  • the CV value was 34%. Therefore, the subsequent culture evaluation was not performed.
  • Example 3 A well plate in which only the concave and convex portions constituting the first pattern were formed without forming the fine concave and convex pattern (second pattern) on the base film in the same procedure as in Example 1, and the same as in Example 1 was prepared. A cell culture vessel was prepared. When the cell culture vessel thus prepared was evaluated for the cell mass size in the same manner as in Example 1, it was extremely difficult to exchange the cell culture solution, and the evaluation could not be performed.
  • the well plate of the present invention is easy to manufacture, and according to the well plate, a cell mass with high size uniformity can be easily obtained. Further, according to the well plate sheet of the present invention, the well plate can be easily manufactured. Furthermore, since the culture method of the present invention uses the well plate, it is possible to easily cultivate cell clusters with high size uniformity. Therefore, the well plate, well plate sheet, and culture method of the present invention can be suitably used for cell culture that will be used more frequently in the future.

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Abstract

The well plate according to the present application is characterized in that the floors of the wells have a raised-and-recessed pattern, the raised-and-recessed pattern being a composite of a first pattern constituted by a combination of raised and recessed parts and a second pattern constituted by a combination of raised and recessed parts smaller than those of the first pattern, and the second pattern being formed on the surface of the first pattern. The well plate sheet according to the present application is characterized by comprising a substrate film which comprises the abovementioned raised-and-recessed pattern in at least a well region on one surface of the substrate film. The culturing method according to the present application is a method of housing and culturing cells, characterized in that the abovementioned well plate is used and the surface fill rate of the first pattern on the well plate is adjusted according to the average diameter of the cell masses to be obtained by culturing.

Description

ウェルプレート、ウェルプレート用シート及び培養方法Well plate, well plate sheet and culture method
　本発明は、ウェルプレート、ウェルプレート用シート及び培養方法に関する。 The present invention relates to a well plate, a well plate sheet and a culture method.
　従来より、化合物の製造や医療分野での使用を目的として、細胞をプレート上で培養することが広く行われている。このような細胞培養において、生体内における状態により近い細胞塊が得られる三次元細胞培養技術についての改良が進んでいる。このような三次元細胞培養技術としては、例えば複数のウェルを有するウェルプレートを用い、各ウェル内で球状の細胞塊を培養する技術等が挙げられる（特開２０１３－２１５１５２号公報参照）。この技術では、細胞が接着し易くかつ細胞増殖を促進する性質を有するコラーゲンを多孔質膜にコーティングしたものを用いる。このコラーゲン積層多孔質膜の表面に付着した細胞は、通常の平面方向への増殖のみならず、高さ方向へも増殖するため、このコラーゲン積層多孔質膜をウェルの底部に配設することで、ウェル内で球状に細胞塊を成長させることができる。 Conventionally, culturing cells on a plate has been widely performed for the purpose of production of compounds and use in the medical field. In such cell culture, improvements are being made on the three-dimensional cell culture technique that can obtain a cell mass closer to the state in the living body. Examples of such a three-dimensional cell culture technique include a technique of culturing a spherical cell mass in each well using a well plate having a plurality of wells (see JP 2013-215152 A). In this technique, a porous membrane coated with collagen having the property of easily adhering to cells and promoting cell growth is used. Since the cells attached to the surface of the collagen laminated porous membrane grow not only in the normal plane direction but also in the height direction, this collagen laminated porous membrane is arranged at the bottom of the well. The cell mass can be grown spherically in the well.
　しかし、上記従来の技術では、ウェルの大きさに合わせたコラーゲン積層多孔質膜を別途ウェルの底部に配設するため、ウェルプレートの製造工程が煩雑となり易く、ウェル底部へのコラーゲン積層多孔質膜の配設が困難になりがちである。また、近年では微小な細胞塊を多数得るためにウェルの微小化が進んでいるが、このような微小なウェルへのコラーゲン積層多孔質膜の積層がさらに困難となるという不都合がある。また、コラーゲン積層多孔質膜は保存時や細胞培養時にコラーゲンが劣化し易いため、三次元培養を行う度にコラーゲン積層多孔質膜を入れ替える必要が生じ、コラーゲン積層多孔質膜の製造及び積層の困難性と相まって、上記ウェルプレートを用いた三次元培養の手順が煩雑となり、微小な細胞塊が容易に得られ難いという不都合もある。また、従来の技術では、得られる細胞塊の大きさもばらつき易い。 However, in the above conventional technique, a collagen laminated porous membrane matching the size of the well is separately provided at the bottom of the well, so that the manufacturing process of the well plate tends to be complicated, and the collagen laminated porous membrane on the well bottom Is likely to be difficult to arrange. In recent years, wells have been miniaturized in order to obtain a large number of minute cell masses. However, there is a disadvantage that it is more difficult to laminate a collagen-laminated porous membrane in such minute wells. In addition, since the collagen-laminated porous membrane is prone to degradation of collagen during storage and cell culture, it is necessary to replace the collagen-laminated porous membrane every time three-dimensional culture is performed, making it difficult to produce and laminate the collagen-laminated porous membrane. Combined with the characteristics, the procedure of three-dimensional culture using the well plate becomes complicated, and there is a disadvantage that it is difficult to easily obtain a minute cell mass. Further, in the conventional technique, the size of the obtained cell mass is likely to vary.
特開２０１３－２１５１５２号公報JP 2013-215152 A
　本発明は以上のような事情に基づいてなされたものであり、その目的は、製造が容易であり、かつサイズの均一性が高い微小な細胞塊を容易に得ることができるウェルプレート、このウェルプレートの製造に用いるウェルプレート用シート、及びこのウェルプレートを用いた培養方法を提供することにある。 The present invention has been made on the basis of the circumstances as described above, and an object of the present invention is to provide a well plate that can be easily manufactured and that can easily obtain a minute cell mass with high size uniformity, and the well. It is an object of the present invention to provide a well plate sheet used for producing a plate and a culture method using the well plate.
　上記課題を解決するためになされた発明は、細胞を収容し、培養する１又は複数のウェルを備えるウェルプレートであって、上記ウェルが底面に凹凸パターンを有し、上記凹凸パターンが、凹凸の組み合わせにより構成される第１パターンと、上記第１パターンよりも微細な凹凸の組み合わせにより構成される第２パターンとの複合パターンであり、上記第１パターンの表面に上記第２パターンが形成されていることを特徴とする。 The invention made to solve the above-mentioned problems is a well plate having one or a plurality of wells for containing and culturing cells, wherein the wells have a concavo-convex pattern on the bottom surface, and the concavo-convex pattern is a concavo-convex pattern. It is a composite pattern of a first pattern constituted by a combination and a second pattern constituted by a combination of unevenness finer than the first pattern, and the second pattern is formed on the surface of the first pattern. It is characterized by being.
　また、上記課題を解決するためになされた別の本発明は、細胞を収容し、培養する１又は複数のウェルを備えるウェルプレートのウェル底面を構成するシートであって、基材フィルムを備え、上記基材フィルムが一方の面のうち少なくとも上記ウェル領域に凹凸パターンを有し、上記凹凸パターンが、凹凸の組み合わせにより構成される第１パターンと、上記第１パターンよりも微細な凹凸の組み合わせにより構成される第２パターンとの複合パターンであり、上記第１パターンの表面に上記第２パターンが形成されていることを特徴とする。 Another aspect of the present invention made to solve the above-described problem is a sheet that forms the well bottom surface of a well plate including one or a plurality of wells that contain and culture cells, and includes a base film, The base film has a concavo-convex pattern in at least the well region of one surface, and the concavo-convex pattern is a combination of a concavo-convex finer than the first pattern, and a first concavo-convex pattern formed by a combination of concavo-convex patterns It is a composite pattern with the 2nd pattern comprised, The said 2nd pattern is formed in the surface of the said 1st pattern, It is characterized by the above-mentioned.
　上記課題を解決するためになされたさらに別の本発明は、細胞を収容し、培養する方法であって、当該ウェルプレートを用い、上記培養で得る細胞塊の平均径に応じて上記ウェルプレートの第１パターンの平面充填率を調整することを特徴とする培養方法である。 Yet another aspect of the present invention made to solve the above-mentioned problems is a method for containing and culturing cells, wherein the well plate is used, and the well plate is subjected to the average diameter of the cell mass obtained by the culture. The culture method is characterized in that the flat filling factor of the first pattern is adjusted.
　当該ウェルプレートは製造が容易であり、かつ当該ウェルプレートによればサイズの均一性が高い細胞塊を容易に得ることができる。また、当該ウェルプレート用シートによれば、容易に当該ウェルプレートを製造することができる。さらに、当該培養方法は、当該ウェルプレートを用いるため、サイズの均一性が高い細胞塊を容易に培養することができる。 The well plate is easy to manufacture, and according to the well plate, a cell mass with high size uniformity can be easily obtained. Further, according to the well plate sheet, the well plate can be easily manufactured. Furthermore, since the culturing method uses the well plate, it is possible to easily cultivate a cell mass having high size uniformity.
　また、当該ウェルプレートは、微細な凹凸を有する第２パターンによって、培養によって生ずる細胞塊が足場を作りウェルプレートに固定しやすくすることができる。 In addition, the well plate can be easily fixed to the well plate by the cell mass generated by the culture by using the second pattern having fine irregularities.
本発明の一実施形態にかかるウェルプレートの模式的平面図である。It is a typical top view of a well plate concerning one embodiment of the present invention. 図１のウェルプレートのＡ－Ａ線における模式的拡大部分断面図である。FIG. 2 is a schematic enlarged partial sectional view taken along line AA of the well plate of FIG. 1. 実施例１のウェルプレートのウェル底面を示すレーザー顕微鏡写真である。2 is a laser micrograph showing the well bottom of the well plate of Example 1. FIG. 実施例１のウェルプレートの微細凹凸パターンを示すレーザー顕微鏡写真である。2 is a laser micrograph showing a fine uneven pattern of a well plate of Example 1. FIG.
　以下、適宜図面を参照しつつ本発明の実施の形態を詳説する。 Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings as appropriate.
＜ウェルプレート＞
　当該ウェルプレートは、図１及び図２に示すように、細胞を収容し、培養する複数のウェル１を備える。このウェル１は底面２と内周面３とからなり、それぞれのウェル１の底面２に凹凸パターンを有する。この凹凸パターンは、凹凸の組み合わせにより構成される第１パターン１ａと、この第１パターン１ａよりも微細な凹凸の組み合わせにより構成され、第１パターン１ａと複合される第２パターン（微細凹凸パターン）１ｂとを有する。具体的には、第１パターン１ａの表面に第２パターン１ｂが形成されており、上記第１パターン１ａの表面に上記第２パターン１ｂが形成されている。なお、「凹凸が微細」とは、凹凸の平均ピッチ及び平均高低差（隣接する谷部の最下点と山部の最上点との平均水平距離及び平均垂直距離）が小さいことを意味する。 <Well plate>
As shown in FIGS. 1 and 2, the well plate includes a plurality of wells 1 for containing and culturing cells. The well 1 includes a bottom surface 2 and an inner peripheral surface 3, and the bottom surface 2 of each well 1 has an uneven pattern. The concavo-convex pattern includes a first pattern 1a configured by a combination of concavo-convex and a second pattern (fine concavo-convex pattern) that is configured by a combination of concavo-convex finer than the first pattern 1a and combined with the first pattern 1a. 1b. Specifically, the second pattern 1b is formed on the surface of the first pattern 1a, and the second pattern 1b is formed on the surface of the first pattern 1a. “Fine unevenness” means that the average pitch and average height difference (average horizontal distance and average vertical distance between the lowest point of the adjacent valley and the highest point of the peak) are small.
　また、当該ウェルプレートは、ウェル１の側壁を構成するウェルプレート本体４、ウェル１の底壁を構成する底面板５、及びウェルプレート本体４と底面板５との間に積層され、ウェル１の底面２を構成するウェルプレート用シート６を主に備える。このウェルプレート本体４は、複数の貫通孔７を有する厚板状である。また、上記第１パターン１ａを構成する凹凸の凹部はウェルプレート用シート６の表面に形成されている。 The well plate is stacked between the well plate body 4 constituting the side wall of the well 1, the bottom plate 5 constituting the bottom wall of the well 1, and the well plate body 4 and the bottom plate 5. A well plate sheet 6 constituting the bottom surface 2 is mainly provided. The well plate body 4 has a thick plate shape having a plurality of through holes 7. Further, the concave and convex concave portions constituting the first pattern 1 a are formed on the surface of the well plate sheet 6.
　当該ウェルプレートの平面視における形状としては、矩形状、円形状等が挙げられるが、矩形状が好ましい。当該ウェルプレートが平面視矩形である場合、その平面方向における短辺及び長辺の長さは、ウェル１の大きさや配設密度、培養に用いる機器等の大きさ等に応じ適宜調整できる。例えば９６、３８４及び１５３６ウェルに関してはＡＮＳＩ／ＳＢＳ　１－２００４～４－２００４の規格がある。 The shape of the well plate in plan view includes a rectangular shape and a circular shape, but a rectangular shape is preferable. When the well plate has a rectangular shape in plan view, the length of the short side and the long side in the plane direction can be adjusted as appropriate according to the size and arrangement density of the wells 1 and the size of the equipment used for culture. For example, for 96, 384 and 1536 wells, there are ANSI / SBS 1-2004 to 4-2004 standards.
　ウェルプレート本体４及び底面板５の主成分としては、培養する細胞に悪影響を与えず、また培養液により変質等しないものであれば特に限定されないが、例えばポリプロピレン、ポリエステル、ポリオレフィン、ポリスチレン、ポリウレタン、セルロース、再生セルロース、ポリカーボネート、ポリアミド、ポリイミド、ポリテトラフルオロエチレン等のフッ素含有樹脂などが挙げられる。ここで「主成分」とは、質量基準で最も多い成分（例えば５０質量％以上）を意味する。また、ウェルプレート本体４及び底面板５は、上記主成分の他に各種添加剤を含有してもよい。 The main component of the well plate body 4 and the bottom plate 5 is not particularly limited as long as it does not adversely affect the cells to be cultured and does not change in quality by the culture solution. For example, polypropylene, polyester, polyolefin, polystyrene, polyurethane, Examples thereof include fluorine-containing resins such as cellulose, regenerated cellulose, polycarbonate, polyamide, polyimide and polytetrafluoroethylene. Here, the “main component” means the most abundant component (for example, 50% by mass or more) on a mass basis. Further, the well plate body 4 and the bottom plate 5 may contain various additives in addition to the main components.
　また、ウェルプレート本体４及び底面板５は、同じ材質であってもよく、異なっていてもよいが、同じ材質であることが好ましい。ウェルプレート本体４及び底面板５が同じ材質であることで、後述する接合工程において、ウェルプレート本体４と底面板５とを容易に接合できる。 Further, the well plate body 4 and the bottom plate 5 may be made of the same material or different, but are preferably made of the same material. Since the well plate body 4 and the bottom plate 5 are made of the same material, the well plate body 4 and the bottom plate 5 can be easily joined in the joining step described later.
　ウェル１の形状としては、当該ウェルプレートの底面に垂直な方向を軸とする角柱状、角錐台状、円柱状、円錐台状、楕円柱状、楕円錐台状等が挙げられる。ここで、当該ウェルプレートの底面とは、「ウェルプレート本体４のうち、ウェルプレート本体４とウェルプレート用シート６とが接触している面」をいう。ウェル１の形状としては、これらの中で円柱状及び円錐台状が好ましく、円錐台状がより好ましく、底面側が小さく開口側が大きい円錐台状がさらに好ましい。すなわち、ウェル１の内径が底面２から開口に向かって漸増することがより好ましい。ウェル１の形状がこのようなものであることで、ウェルプレート本体４を射出成形する場合に金型からウェルプレートを容易に外すことができ、当該ウェルプレートの製造がより容易となる。また、開口が相対的に大きいため、ウェル１内への培養液の導入や、細胞培養中の各種操作も容易となる。 Examples of the shape of the well 1 include a prismatic shape, a truncated pyramid shape, a cylindrical shape, a truncated cone shape, an elliptical columnar shape, and an elliptical truncated cone shape whose axis is a direction perpendicular to the bottom surface of the well plate. Here, the bottom surface of the well plate means “the surface of the well plate body 4 where the well plate body 4 and the well plate sheet 6 are in contact”. Of these, the shape of the well 1 is preferably a columnar shape and a truncated cone shape, more preferably a truncated cone shape, and more preferably a truncated cone shape having a small bottom side and a large opening side. That is, it is more preferable that the inner diameter of the well 1 gradually increases from the bottom surface 2 toward the opening. When the well 1 has such a shape, the well plate can be easily removed from the mold when the well plate body 4 is injection-molded, and the manufacture of the well plate becomes easier. In addition, since the opening is relatively large, introduction of the culture medium into the well 1 and various operations during cell culture are facilitated.
　また、それぞれのウェル１の底面直径Ｄ１の下限としては、０．５ｍｍが好ましく、０．８ｍｍがより好ましく、１ｍｍがさらに好ましい。一方、上記底面直径Ｄ１の上限としては、１０ｃｍが好ましく、５ｃｍがより好ましく、３．５ｃｍがさらに好ましい。上記直径Ｄ１が上記下限より小さいと、ウェル１内への培養液の導入が困難となるおそれがある。逆に、上記底面直径Ｄ１が上記上限を超えると、三次元培養可能な細胞塊の大きさに対してウェル１が過剰に大きくなり、細胞培養に寄与しないスペースが増加する結果、当該ウェルプレートを用いて三次元培養を行う際の培養効率が低下するおそれがある。なお、ウェル１の断面が楕円である場合のウェル１の底面直径Ｄ１とは、長径及び短径の平均値を意味し、ウェル１の断面が矩形である場合のウェル１の底面直径Ｄ１とは、長辺及び短辺の平均値を意味する。 Also, the lower limit of the bottom surface diameter D1 of each well 1 is preferably 0.5 mm, more preferably 0.8 mm, and even more preferably 1 mm. On the other hand, the upper limit of the bottom diameter D1 is preferably 10 cm, more preferably 5 cm, and even more preferably 3.5 cm. If the diameter D1 is smaller than the lower limit, introduction of the culture medium into the well 1 may be difficult. Conversely, if the bottom diameter D1 exceeds the upper limit, the well 1 becomes excessively large relative to the size of the cell mass that can be three-dimensionally cultured, and as a result, the space that does not contribute to cell culture increases. There is a possibility that the culture efficiency when performing the three-dimensional culture by using it decreases. The bottom diameter D1 of the well 1 when the cross section of the well 1 is an ellipse means the average value of the major axis and the minor axis, and the bottom diameter D1 of the well 1 when the cross section of the well 1 is a rectangle. Mean the average value of the long and short sides.
　ウェル１の断面形状が楕円である場合における扁平率の上限としては、０．２が好ましく、０．１５がより好ましい。上記扁平率が上記上限を超えると、細胞培養に寄与しないスペースが増加し培養効率が低下するおそれや、得られる細胞塊の形状における対称性が低下し、均質な細胞塊を多数得ることが困難となるおそれがある。ここで「扁平率」とは、楕円の長軸半径をａ、短軸半径をｂとした場合に、（ａ－ｂ）／ａで表される値を意味する。 When the cross-sectional shape of the well 1 is an ellipse, the upper limit of the flatness is preferably 0.2 and more preferably 0.15. If the flatness exceeds the above upper limit, the space that does not contribute to cell culture increases and the culture efficiency may decrease, or the symmetry of the shape of the resulting cell mass decreases, making it difficult to obtain many homogeneous cell masses. There is a risk of becoming. Here, the “flattening ratio” means a value represented by (ab) / a where the major axis radius of the ellipse is a and the minor axis radius is b.
　また、ウェル１の断面形状が矩形である場合、その短辺に対する長辺の比の下限としては、１が好ましい。一方、上記比の上限としては、１．３が好ましく、１．２がより好ましい。上記比が上記上限を超えると、細胞培養に寄与しないスペースが増加し培養効率が低下するおそれや、得られる細胞塊の形状における対称性が低下し、均質な細胞塊を多数得ることが困難となるおそれがある。 Further, when the cross-sectional shape of the well 1 is rectangular, the lower limit of the ratio of the long side to the short side is preferably 1. On the other hand, the upper limit of the ratio is preferably 1.3 and more preferably 1.2. If the ratio exceeds the above upper limit, the space that does not contribute to cell culture increases and the culture efficiency may decrease, the symmetry in the shape of the resulting cell mass decreases, and it is difficult to obtain a large number of homogeneous cell masses There is a risk.
　それぞれのウェル１の深さの下限としては、０．５ｍｍが好ましく、１ｍｍがより好ましく、１．２ｍｍがさらに好ましい。一方、上記深さの上限としては、２ｃｍが好ましく、１．８ｃｍがより好ましく、１．５ｃｍがさらに好ましい。上記深さが上記下限より小さいと、ウェル１内に安定して培養液を保持し難くなり、細胞の三次元培養が困難となるおそれがある。逆に、上記深さが上記上限を超えると、培養後の細胞塊をウェル１から取り出し難くなるおそれがある。 The lower limit of the depth of each well 1 is preferably 0.5 mm, more preferably 1 mm, and even more preferably 1.2 mm. On the other hand, the upper limit of the depth is preferably 2 cm, more preferably 1.8 cm, and even more preferably 1.5 cm. When the depth is smaller than the lower limit, it is difficult to stably hold the culture solution in the well 1 and it may be difficult to perform three-dimensional culture of cells. On the other hand, when the depth exceeds the upper limit, it is difficult to take out the cell mass after culturing from the well 1.
　それぞれのウェル１の容積の下限としては、０．１μＬが好ましく、０．５μＬがより好ましく、０．８μＬがさらに好ましい。一方、上記容積の上限としては、１０ｍＬが好ましく、７ｍＬがより好ましく、４ｍＬがさらに好ましい。上記容積が上記下限より小さいと、ウェル１内への培養液の導入が困難となるおそれがある。逆に、上記直径が上記上限を超える場合、三次元培養可能な細胞塊の大きさに対してウェル１が過剰に大きくなり、使用する培養液の量が不必要に増加し、三次元培養に伴うコストが増加するおそれがある。 The lower limit of the volume of each well 1 is preferably 0.1 μL, more preferably 0.5 μL, and even more preferably 0.8 μL. On the other hand, the upper limit of the volume is preferably 10 mL, more preferably 7 mL, and even more preferably 4 mL. If the volume is smaller than the lower limit, introduction of the culture solution into the well 1 may be difficult. On the contrary, when the diameter exceeds the upper limit, the well 1 becomes excessively large relative to the size of the cell mass that can be three-dimensionally cultured, and the amount of the culture medium to be used is unnecessarily increased. The associated costs may increase.
　また、複数のウェル１の形状は同じであることが好ましい。このように、当該ウェルプレートが同じ形状のウェル１を複数備えることで、形状の均質性に優れた複数の細胞塊を一度の培養手順により多数得ることができる。 Moreover, it is preferable that the shapes of the plurality of wells 1 are the same. As described above, when the well plate includes a plurality of wells 1 having the same shape, a large number of cell clusters having excellent shape homogeneity can be obtained by a single culture procedure.
（第１パターン）
　それぞれのウェル１の底面２は、第１パターン１ａを構成する複数の凹部を有し、この凹部は表面（内面）全体に微細な凹凸パターンである第２パターン１ｂを有する。このようにウェル１の底面２に第２パターン１ｂが形成された凹部を有することで、培養液中に散布された細胞が上記第１パターン１ａを構成する凹凸の凹部中に集まることで均一な大きさ（ＣＶ値の小さい）の細胞塊を得ることができる。また、上記凹部の表面に形成された第２パターン１ｂにより、播種された細胞が多くの仮足を成長させるようになり、ウェル１への接着性が向上する。この細胞は上記凹部の表面（内面）に沿うようにして平面方向に成長すると共に、上記凹部内で成長した部分を足場として、その後も高さ方向に成長を続けるため、効率よく細胞塊を得ることができる。 (First pattern)
The bottom surface 2 of each well 1 has a plurality of recesses constituting the first pattern 1a, and this recess has a second pattern 1b which is a fine uneven pattern on the entire surface (inner surface). Thus, by having the recessed part in which the 2nd pattern 1b was formed in the bottom face 2 of the well 1, the cell spread | dispersed in the culture solution gathered in the uneven recessed part which comprises the said 1st pattern 1a, and is uniform. A cell cluster having a size (small CV value) can be obtained. In addition, the second pattern 1b formed on the surface of the concave portion allows the seeded cells to grow many provisional limbs, and the adhesion to the well 1 is improved. This cell grows in a planar direction along the surface (inner surface) of the recess, and continues to grow in the height direction using the portion grown in the recess as a scaffold, so that a cell mass is efficiently obtained. be able to.
　また、この第１パターン１ａ及び第２パターン１ｂの複合凹凸パターンは、一度底面２に形成された後は底面２の一部として存在し、かつ保存や細胞培養時に劣化し難い。そのため、ウェルの底面２のメンテナンス等が容易となり、その結果簡便な手順で細胞を三次元培養できる。 Further, the composite uneven pattern of the first pattern 1a and the second pattern 1b exists as a part of the bottom surface 2 once formed on the bottom surface 2, and hardly deteriorates during storage or cell culture. Therefore, maintenance of the bottom surface 2 of the well is facilitated, and as a result, the cells can be three-dimensionally cultured by a simple procedure.
　さらに、この複合凹凸パターンは、ウェルプレート本体４と接合する前の底面板５やウェルプレート用シート６において形成することが可能であり、微細なウェル１内で第１パターン１ａを構成する凹凸の凹部の形成作業を行う必要がない。そのため、当該ウェルプレートは製造が容易である。 Furthermore, this composite uneven pattern can be formed on the bottom plate 5 and the well plate sheet 6 before being joined to the well plate body 4, and the uneven patterns constituting the first pattern 1a in the fine well 1 can be formed. There is no need to perform the operation of forming the recess. Therefore, the well plate is easy to manufacture.
　上記第１パターン１ａを構成する凹凸の凹部の平面視における形状としては、円、楕円、矩形等の多角形などとすることができるが、円形であることが好ましい。このように、上記凹部が円形であることで、培養される細胞塊の形状をより球に近づけることができ、その結果形状が均質な細胞塊を容易に得ることができる。 The shape of the concave / convex concave portions constituting the first pattern 1a can be a polygon such as a circle, an ellipse, or a rectangle, but is preferably a circle. Thus, since the concave portion is circular, the shape of the cultured cell mass can be made closer to a sphere, and as a result, a homogeneous cell mass can be easily obtained.
　それぞれのウェル１内の１の第１パターン１ａを構成する凹凸の凹部の平均径Ｄ２の下限としては、３０μｍが好ましく、１００μｍがより好ましく、１５０μｍがさらに好ましい。一方、上記平均径Ｄ２の上限としては、３０００μｍが好ましく、２０００μｍがより好ましく、１０００μｍがさらに好ましい。上記平均径Ｄ２が上記下限より小さいと、得られる細胞塊の大きさが過度に小さくなるおそれがある。逆に、上記平均径Ｄ２が上記上限を超えると、三次元培養時に細胞培養に寄与しないスペースが大きくなり、細胞の培養効率が低下するおそれや、細胞が平面的に過度に成長する結果三次元培養し難くなるおそれがある。ここで「凹部の平均径」とは、凹部の平面視面積と同面積の真円の直径の平均値を意味する。 The lower limit of the average diameter D2 of the concave and convex portions constituting one first pattern 1a in each well 1 is preferably 30 μm, more preferably 100 μm, and even more preferably 150 μm. On the other hand, the upper limit of the average diameter D2 is preferably 3000 μm, more preferably 2000 μm, and even more preferably 1000 μm. If the average diameter D2 is smaller than the lower limit, the size of the obtained cell mass may be excessively small. On the other hand, if the average diameter D2 exceeds the upper limit, the space that does not contribute to cell culture during 3D culture increases, and the cell culture efficiency may decrease, or the cells may grow excessively in a plane. May be difficult to culture. Here, the “average diameter of the recesses” means an average value of the diameters of perfect circles having the same area as the planar view area of the recesses.
　それぞれのウェル１内の第１パターン１ａを構成する凹凸の凹部の平面充填率（第１パターンの平面充填率）は培養を行う際の細胞種や播種数によって異なるが、上記平面充填率の下限としては、５％が好ましく、１０％がより好ましい。一方、上記平面充填率の上限としては、９９．９％が好ましく、９０％がより好ましい。上記平面充填率が上記下限より小さいと、播種数が増えると凹部に入らない細胞が増えてしまうため、細胞塊のＣＶ値（バラツキ）が大きくなるおそれがある。逆に、上記平面充填率が上記上限を超えると、第１パターン１ａを安定的に成形することが難しくなる。ここで「凹部の平面充填率」とは、１つのウェルの底面の面積に対するこのウェル内の凹部の平面視面積の合計の比を意味する。 The plane filling rate of the concave and convex recesses constituting the first pattern 1a in each well 1 (planar filling rate of the first pattern) varies depending on the cell type and the number of seeding when culturing, but the lower limit of the plane filling rate. Is preferably 5%, more preferably 10%. On the other hand, the upper limit of the plane filling rate is preferably 99.9% and more preferably 90%. If the plane filling rate is smaller than the lower limit, the number of cells that do not enter the recess increases as the number of seeding increases, which may increase the CV value (variation) of the cell mass. On the other hand, when the plane filling rate exceeds the upper limit, it is difficult to stably mold the first pattern 1a. Here, the “planar filling ratio of the recesses” means the ratio of the total area in plan view of the recesses in the wells to the area of the bottom surface of one well.
　第１パターン１ａを構成する凹凸の凹部の形状としては、製造性と細胞の培養性との観点から、当該ウェルプレートの底面に垂直な方向を軸とする立体形状であることが好ましく、角柱状、角錐台状、円柱状、円錐台状、楕円柱状、楕円錐台状等の平底の有底筒状、角錐状、円錐状、半球状、半楕円球状がさらに好ましく、特に半球状（レンズ状）又は平底の有底筒状が好ましい。 The shape of the concave and convex recesses constituting the first pattern 1a is preferably a three-dimensional shape with the direction perpendicular to the bottom surface of the well plate as an axis, from the viewpoints of manufacturability and cell culture. Further, a flat bottomed cylindrical shape such as a truncated pyramid shape, a cylindrical shape, a truncated cone shape, an elliptical columnar shape, an elliptical truncated cone shape, a pyramidal shape, a conical shape, a hemispherical shape, and a semi-elliptical spherical shape are more preferable. ) Or a flat bottomed cylindrical shape.
　第１パターン１ａを構成する凹凸の凹部の最大深さの下限としては、２０μｍが好ましく、５０μｍがより好ましい。一方、上記最大深さの上限としては、２０００μｍが好ましく、１０００μｍがより好ましい。上記最大深さが上記下限より小さいと、上記凹部による三次元培養効率の向上効果が不十分となるおそれがある。逆に、上記最大深さが上記上限を超えると、培養後の細胞を上記凹部内から取り出し難くなるおそれがある。 The lower limit of the maximum depth of the concave and convex portions constituting the first pattern 1a is preferably 20 μm, and more preferably 50 μm. On the other hand, the upper limit of the maximum depth is preferably 2000 μm, and more preferably 1000 μm. If the maximum depth is smaller than the lower limit, the effect of improving the three-dimensional culture efficiency by the recesses may be insufficient. Conversely, if the maximum depth exceeds the upper limit, it may be difficult to take out the cultured cells from the recess.
　第１パターン１ａを構成する凹凸の凹部が半球状である場合、その曲率半径の下限としては、１０μｍが好ましく、２５μｍがより好ましい。一方、上記曲率半径の上限としては、１０００μｍが好ましく、５００μｍがより好ましい。上記曲率半径が上記下限より小さいと、培養後の細胞を上記凹部内から取り出しにくくなるおそれがある。一方、上記曲率半径が上記上限を超えると、上記凹部の平均径に対し深さが過度に小さくなり、上記凹部による三次元培養効率の向上効果が不十分となるおそれがある。 When the concave and convex portions constituting the first pattern 1a are hemispherical, the lower limit of the radius of curvature is preferably 10 μm, and more preferably 25 μm. On the other hand, the upper limit of the radius of curvature is preferably 1000 μm, and more preferably 500 μm. If the curvature radius is smaller than the lower limit, it may be difficult to take out the cultured cells from the recess. On the other hand, when the curvature radius exceeds the upper limit, the depth becomes excessively small with respect to the average diameter of the recess, and the effect of improving the three-dimensional culture efficiency by the recess may be insufficient.
　１つのウェル１が複数の第１パターン１ａを構成する凹凸の凹部を有する場合、これらの凹部は規則的に配列しているとよい。つまり、第１パターン１ａが規則的パターンであるとよい。このように、複数の上記凹部が規則的に配列することで、複数の細胞塊を培養する場合において、それぞれの細胞塊が等間隔に配列する。そのため、隣接する細胞塊の接触及び融合による細胞塊の大きさの予期しない変化を低減できる。その結果、得られる細胞塊の質の均一性をより向上できる。 When one well 1 has concave and convex portions constituting a plurality of first patterns 1a, these concave portions may be regularly arranged. That is, the first pattern 1a is preferably a regular pattern. As described above, when the plurality of concave portions are regularly arranged, when the plurality of cell masses are cultured, the respective cell masses are arranged at equal intervals. Therefore, an unexpected change in the size of the cell mass due to contact and fusion between adjacent cell masses can be reduced. As a result, the uniformity of the quality of the obtained cell mass can be further improved.
　また、１つのウェル１が複数の第１パターン１ａを構成する凹凸の凹部を有する場合、これらの凹部が平面視で単一の形状を有するとよい。このように複数の上記凹部が平面視で単一の形状を有することで、それぞれの上記凹部において培養される細胞塊の形状をより均一化できる。その結果、得られる細胞塊の質の均一性をさらに向上できる。 In addition, in the case where one well 1 has concave and convex concave portions constituting a plurality of first patterns 1a, these concave portions may have a single shape in plan view. Thus, since the said several recessed part has a single shape by planar view, the shape of the cell mass cultured in each said recessed part can be made more uniform. As a result, the uniformity of the quality of the obtained cell mass can be further improved.
　また、上記複数の第１パターン１ａを構成する凹凸の凹部の断面形状が単一の形状であるとよい。このように複数の上記凹部の断面形状が単一の形状であることで、それぞれの第１パターン１ａを構成する凹凸の凹部において培養される細胞塊の形状をより均一化できる。その結果、得られる細胞塊の質の均一性をさらに向上できる。 Further, it is preferable that the cross-sectional shape of the concave and convex portions constituting the plurality of first patterns 1a is a single shape. Thus, the shape of the cell mass cultured in the uneven | corrugated recessed part which comprises each 1st pattern 1a can be made more uniform because the cross-sectional shape of several said recessed part is a single shape. As a result, the uniformity of the quality of the obtained cell mass can be further improved.
（第２パターン）
　第２パターン１ｂは、第１パターン１ａを構成する凹凸の凹部よりも微細な凹領域と凸領域とから構成され、その凹領域及び凸領域の幅及び間隔がそれぞれ１０μｍ以下、深さが１０ｎｍ以上のパターンである。第２パターン１ｂの具体的な形状は特に限定されないが、例えば凹領域及び凸領域の平面視形状は円又は多角形とすることができ、全体の平面視形状としては格子形状やハニカム形状が好適に使用できる。 (Second pattern)
The second pattern 1b is composed of a concave region and a convex region that are finer than the concave and convex portions constituting the first pattern 1a, and the width and interval of the concave region and the convex region are each 10 μm or less, and the depth is 10 nm or more. Pattern. The specific shape of the second pattern 1b is not particularly limited. For example, the planar shape of the concave region and the convex region can be a circle or a polygon, and the lattice shape or honeycomb shape is preferable as the overall planar shape. Can be used for
　第２パターン１ｂを構成する凹凸の凹部の形状としては、製造性と細胞の培養性との観点から、半球状（レンズ状）又は平底の有底筒状が好ましい。 The shape of the concave-convex recesses constituting the second pattern 1b is preferably a hemispherical (lens shape) or flat bottomed cylindrical shape from the viewpoints of manufacturability and cell culture.
　それぞれのウェル１内の第２パターン１ｂを構成する凹凸の凹部の平面充填率（第２パターンの平面充填率）は、培養を行う際の細胞種などによって異なるが、この下限としては、１％が好ましく、２０％がより好ましい。一方、上記平面充填率の上限としては、９９％が好ましく、８５％がより好ましい。上記平面充填率が上記範囲外であると、細胞塊の接着性が悪くなり、細胞培養が難しくなるおそれがある。 The planar filling rate of the concave and convex portions constituting the second pattern 1b in each well 1 (planar filling rate of the second pattern) varies depending on the cell type at the time of culturing, but the lower limit is 1% Is preferable, and 20% is more preferable. On the other hand, the upper limit of the plane filling rate is preferably 99%, more preferably 85%. When the plane filling rate is out of the above range, the adhesion of the cell mass is deteriorated and cell culture may be difficult.
　第２パターン１ｂを構成する凹凸の１の凹部の平面視面積は、培養を行う際の細胞種などによって異なるが、この上限としては、１００μｍ２が好ましく、５０μｍ２がより好ましい。これより平面視面積が大きい場合、細胞塊の接着性が悪くなるおそれがある。一方、上記平面視面積の下限としては、１μｍ２が好ましく、２μｍ２がより好ましい。これより平面視面積が小さい場合は、第２パターン１ｂを安定的に形成することが難しい。 Plan view area of one of the recesses of the irregularities constituting the second pattern 1b varies depending on the cell types at the time of carrying out the cultivation, as the upper limit, preferably 100 [mu] m 2, 50 [mu] m 2 is more preferable. If the area in plan view is larger than this, the adhesion of the cell mass may be deteriorated. On the other hand, the lower limit of the plan view area, preferably 1 [mu] m 2, 2 [mu] m 2 is more preferable. If the area in plan view is smaller than this, it is difficult to stably form the second pattern 1b.
　複数の第２パターン１ｂを構成する凹凸の凹部は規則的に配列しているとよい。つまり、第２パターン１ｂが規則的パターンであるとよい。また、これらの凹部が平面視で単一の形状を有するとよい。このようにすることで、得られる細胞塊の質の均一性をさらに向上できる。 It is preferable that the concave and convex concave portions constituting the plurality of second patterns 1b are regularly arranged. That is, the second pattern 1b is preferably a regular pattern. Moreover, it is good for these recessed parts to have a single shape by planar view. By doing in this way, the uniformity of the quality of the obtained cell mass can further be improved.
　上記第２パターン１ｂの成形方法は特に限定されないが、例えばサンドブラスト、エッチング、イオンビーム、放電、レーザー、ナノインプリント、エンボス、射出成形、スクリーン印刷、オフセット印刷、自己組織化技術等を用いて形成することができ、好ましくはナノインプリント技術を用いる。 The method for forming the second pattern 1b is not particularly limited. For example, the second pattern 1b may be formed using sandblasting, etching, ion beam, electric discharge, laser, nanoimprint, embossing, injection molding, screen printing, offset printing, self-organization technology, or the like. Preferably, nanoimprint technology is used.
　１つの第１パターン１ａを構成する凹凸の凹部は、その表面にパターンの異なる複数種の第２パターン１ｂを有するとよい。さらに、これらの複数種の第２パターン１ｂがそれぞれ規則的であるとよい。これにより、細胞のウェル１への接着性をさらに向上できる。 The concave and convex recesses constituting one first pattern 1a may have a plurality of types of second patterns 1b having different patterns on the surface thereof. Further, the plurality of types of second patterns 1b are preferably regular. Thereby, the adhesiveness to the well 1 of a cell can further be improved.
　なお、図２に示すように、第２パターン１ｂは、ウェル１の底面２における第１パターン１ａを構成する凹凸の凹部以外の部分（平坦部）に形成されていてもよいが、細胞が上記凹部からはみ出ないようにするため上記凹部の底面部分のみに形成する（第１パターン１ａを構成する凹凸の凹部の側面や外側には形成しない）ことが好ましい。 As shown in FIG. 2, the second pattern 1b may be formed in a portion (flat portion) other than the concave and convex portions constituting the first pattern 1a on the bottom surface 2 of the well 1, but the cells are In order not to protrude from the recess, it is preferably formed only on the bottom surface portion of the recess (not formed on the side surface or outside of the recess of the unevenness constituting the first pattern 1a).
＜ウェルプレート用シート＞
　当該ウェルプレート用シート６は、図２に示すように、細胞を収容し、三次元培養する複数のウェル１を備えるウェルプレートのウェル底面２を構成するシートであって、基材フィルムを備え、上記基材フィルムの一方の面のうち少なくとも上記ウェル１に対応するウェル領域に上記第１パターン１ａと上記第２パターン１ｂとが複合した凹凸パターンを有する。 <Well plate sheet>
As shown in FIG. 2, the well plate sheet 6 is a sheet that forms a well bottom surface 2 of a well plate that includes a plurality of wells 1 that contain cells and are three-dimensionally cultured, and includes a base film, It has the uneven | corrugated pattern which the said 1st pattern 1a and the said 2nd pattern 1b compounded in the well area | region corresponding to the said well 1 at least among the one surfaces of the said base film.
　当該ウェルプレート用シート６は当該ウェルプレートのウェル領域に上記第１パターン１ａと上記第２パターン１ｂとが複合した凹凸パターンが形成されるため、当該ウェルプレート用シート６を底面板５のウェルプレート本体４が接合される側の面に積層し、その後貫通孔７内に第１パターン１ａを構成する凹凸の凹部が露出するように当該ウェルプレート用シート６の表面にウェルプレート本体４を接合することで、ウェル１の底面２に上記凹部を有する当該ウェルプレートを容易に製造できる。 Since the concave / convex pattern in which the first pattern 1 a and the second pattern 1 b are combined is formed in the well region of the well plate, the well plate sheet 6 is used as the well plate of the bottom plate 5. The well plate main body 4 is laminated on the surface to which the main body 4 is bonded, and then the well plate main body 4 is bonded to the surface of the well plate sheet 6 so that the concave and convex portions constituting the first pattern 1a are exposed in the through holes 7. Thus, the well plate having the concave portion on the bottom surface 2 of the well 1 can be easily manufactured.
　また、当該ウェルプレート用シート６では、シートの大きさをウェル１に合わせる代わりに、ウェル１の大きさに合わせた第１パターン１ａを構成する凹凸の凹部をウェルプレートの大きさに合わせたシート表面に形成することが可能であるため、ウェル１が微小な場合であっても当該ウェルプレートを容易に製造できる。 In the well plate sheet 6, instead of adjusting the size of the sheet to the well 1, the concave and convex portions constituting the first pattern 1a adjusted to the size of the well 1 are adjusted to the size of the well plate. Since it can be formed on the surface, the well plate can be easily manufactured even when the well 1 is very small.
　当該ウェルプレート用シート６の主成分としては、細胞塊の成長を阻害しないものであれば特に限定されないが、上記ウェル１の主成分として挙げたものと同様の樹脂等を用いることができる。これらの中で、ポリオレフィンが好ましく、環状オレフィンポリマーがより好ましい。このような環状オレフィンポリマーは、細胞培養において細胞の成長を阻害せず、かつ微細凹凸パターン（第２パターン）を容易に形成できる。このような環状オレフィンポリマーとしては、例えばＡＲＴＯＮ（ＪＳＲ社製）、ＺＥＯＮＯＲ（日本ゼオン社製）等が挙げられる。 The main component of the well plate sheet 6 is not particularly limited as long as it does not inhibit the growth of cell mass, but the same resin as the main component of the well 1 can be used. Among these, polyolefin is preferable, and cyclic olefin polymer is more preferable. Such a cyclic olefin polymer does not inhibit cell growth in cell culture, and can easily form a fine uneven pattern (second pattern). Examples of such a cyclic olefin polymer include ARTON (manufactured by JSR Corporation), ZEONOR (manufactured by Nippon Zeon Corporation), and the like.
［ウェルプレートの製造方法］
　当該ウェルプレートの製造方法は、特に限定されることはなく、例えば、ウェルプレート本体４、底面板５、及びウェルプレート用シート６を別に作製し接合する方法や、ウェルプレート用シート６上にウェルプレート本体４を直接成形するインサート成形などが挙げられる。この中でも、ウェルプレート本体４、底面板５、及びウェルプレート用シート６を別に作製し接合する方法が好ましい。この方法では、ウェルプレート用シート６上の微細凹凸パターンへのダメージを抑制してウェルプレートを作製できる。具体的には、この方法は、ウェルプレート用シート形成工程、ウェルプレート本体成形工程、及び積層工程を備える。 [Manufacturing method of well plate]
The manufacturing method of the well plate is not particularly limited. For example, the well plate body 4, the bottom plate 5, and the well plate sheet 6 are separately manufactured and bonded, or the well plate is formed on the well plate sheet 6. Examples include insert molding that directly molds the plate body 4. Among these, a method in which the well plate body 4, the bottom plate 5, and the well plate sheet 6 are separately manufactured and bonded is preferable. In this method, the well plate can be produced while suppressing damage to the fine uneven pattern on the well plate sheet 6. Specifically, this method includes a well plate sheet forming step, a well plate body forming step, and a laminating step.
（ウェルプレート用シート形成工程）
　ウェルプレート用シート６の形成方法は特に限定されないが、上記第２パターン１ｂの説明で例示したエッチング等の方法により第１パターン１ａを構成する凹凸の凹部及び第２パターン１ｂを形成する方法が挙げられる。好ましくは、一方の面に第２パターン１ｂを作製した基材フィルムに、上記凹部のパターンを有する金型を第２パターン１ｂの反対面より当てて第１パターン１ａを構成する凹凸の凹部を形成することでウェルプレート用シート６が得られる。この方法では、第２パターン１ｂを安定的にフィルム上に形成することができる。 (Well plate sheet forming process)
The method for forming the well plate sheet 6 is not particularly limited, and examples thereof include a method for forming the concave and convex concave portions and the second pattern 1b constituting the first pattern 1a by the method such as etching exemplified in the description of the second pattern 1b. It is done. Preferably, a concave / convex concave portion constituting the first pattern 1a is formed on the base film on which the second pattern 1b is formed on one surface by applying a mold having the concave pattern from the opposite surface of the second pattern 1b. Thus, the well plate sheet 6 is obtained. In this method, the second pattern 1b can be stably formed on the film.
　なお、上述の方法において、ウェルプレート用シート６のウェル領域以外の領域にも凹凸パターンを形成してもよい。ウェル領域外の凹凸パターンは、後述するウェルプレート本体４の接合により埋められる。 In the above-described method, an uneven pattern may be formed in a region other than the well region of the well plate sheet 6. The concavo-convex pattern outside the well region is filled by bonding of the well plate body 4 described later.
（ウェルプレート本体成形工程）
　ウェルプレート本体成形工程では、複数の貫通孔７を有するウェルプレート本体４を樹脂成形する。この貫通孔７の内周面は、ウェル１の内周面３に相当する。 (Well plate body molding process)
In the well plate body molding step, the well plate body 4 having a plurality of through holes 7 is molded with resin. The inner peripheral surface of the through hole 7 corresponds to the inner peripheral surface 3 of the well 1.
　上記樹脂成形の方法としては公知の方法を採用でき、例えば樹脂板をプレス成形する方法、金型に液状の樹脂を流し込み硬化させる方法、金型に樹脂を射出成形する方法等が挙げられ、これらの中で射出成形する方法が好ましい。樹脂成形の方法として射出成形する方法を用いることで、貫通孔７を有するウェルプレート本体４を容易に成形できる。 As the resin molding method, a known method can be adopted, for example, a method of press molding a resin plate, a method of pouring a liquid resin into a mold and curing it, a method of injection molding of a resin into a mold, etc. Of these, the method of injection molding is preferred. By using the injection molding method as the resin molding method, the well plate body 4 having the through holes 7 can be easily molded.
（積層工程）
　積層工程では、ウェルプレート本体４とウェルプレート用シート６と底面板５とを積層し、これらを接合する。底面板５にウェルプレート用シート６が積層されている場合、底面板５及びウェルプレート用シート６の積層体のウェルプレート用シート６側の面にウェルプレート本体４を接合する。 (Lamination process)
In the laminating step, the well plate body 4, the well plate sheet 6, and the bottom plate 5 are laminated and joined together. When the well plate sheet 6 is laminated on the bottom plate 5, the well plate body 4 is joined to the surface on the well plate sheet 6 side of the laminate of the bottom plate 5 and the well plate sheet 6.
　積層工程におけるウェルプレート本体４とウェルプレート用シート６又は上記積層体との接合方法としては公知の方法を採用でき、例えば接着剤やテープを用いた接着、熱プレス等による圧着、超音波やレーザーによる溶融接着などが挙げられる。これらの中で、熱プレス等による圧着、超音波やレーザーによる溶融接着が好ましい。接合方法としてこれらの方法を用いることで、細胞培養時における接着剤等の成分の培養液への溶出を防止することができる。 As a method for joining the well plate body 4 and the well plate sheet 6 or the laminated body in the laminating process, a known method can be adopted, for example, adhesion using an adhesive or a tape, pressure bonding by a hot press, ultrasonic wave or laser. For example, melt adhesion by the above. Among these, pressure bonding by hot press or the like, and melt bonding by ultrasonic waves or lasers are preferable. By using these methods as joining methods, it is possible to prevent elution of components such as adhesives into the culture solution during cell culture.
＜培養方法＞
　当該培養方法は、当該ウェルプレートを用い、培養で得る細胞塊の平均径に応じて上記ウェルプレートの凹部の平面充填率を調整することを特徴とする。 <Culture method>
The culture method is characterized by using the well plate and adjusting the plane filling rate of the recesses of the well plate according to the average diameter of the cell mass obtained by the culture.
　当該培養方法では、当該ウェルプレートを用いるため、上述のようにウェル底面の凹部内において細胞が三次元成長する。そのため、三次元培養で得る細胞塊の平均径に応じてウェルプレートの凹部（第１パターン）の平面充填率を調整することで、所望の大きさの細胞塊を容易に得ることができる。 In the culturing method, since the well plate is used, the cells grow three-dimensionally in the recesses on the bottom surface of the well as described above. Therefore, by adjusting the plane filling rate of the recesses (first pattern) of the well plate according to the average diameter of the cell mass obtained by three-dimensional culture, a cell mass of a desired size can be easily obtained.
　ここで、三次元培養で得る細胞塊のバラツキについてはＣＶ値で表すことができる。ＣＶ値は、細胞塊の大きさの標準偏差σを平均値で割った値（％）である。ＣＶ値が小さいほど、細胞塊の均一性は高いといえる。このＣＶ値は凹部の平面視充填率によって変化する。 Here, the variation of the cell mass obtained by the three-dimensional culture can be expressed by a CV value. The CV value is a value (%) obtained by dividing the standard deviation σ of the cell mass size by the average value. It can be said that the smaller the CV value, the higher the uniformity of the cell mass. This CV value varies depending on the filling ratio of the concave portion in plan view.
　当該培養方法における細胞の培養手順としては、公知のものを採用することができる。具体的には、当該ウェルプレートの各ウェル内へ培養液を導入する工程、培養する細胞を各ウェル内へ散布する工程、及び各ウェル内で細胞を培養する工程を備えるものが挙げられる。 As a cell culturing procedure in the culturing method, a known one can be adopted. Specific examples include a step of introducing a culture solution into each well of the well plate, a step of dispersing cells to be cultured into each well, and a step of culturing the cells in each well.
［その他の実施形態］
　当該ウェルプレート、ウェルプレート用シート及び培養方法は、上記実施形態に限定されるものではない。上記実施形態では、第１パターンが凹部を有する形態としたが、凹部の代わりに複数の凸部を有し、この凸部の表面（外面）に第２パターンを形成してもよい。また、上記凹部の表面に凹部分のみからなる第２パターンを形成してもよいし、上記凸部の表面に凸部分のみからなる第２パターンを形成してもよい。 [Other Embodiments]
The well plate, the well plate sheet and the culture method are not limited to the above embodiment. In the said embodiment, although the 1st pattern was set as the form which has a recessed part, it may have a some convex part instead of a recessed part, and may form a 2nd pattern in the surface (outer surface) of this convex part. Moreover, the 2nd pattern which consists only of a recessed part may be formed in the surface of the said recessed part, and the 2nd pattern which consists only of a convex part may be formed in the surface of the said convex part.
　当該ウェルプレートが備えるウェルの数は１でもよい。また、第１パターンを構成する凹凸の凹部又は凸部は上記実施形態のように１つのウェルにつき１つのみ形成されてもよく、１つのウェルにつき複数形成されてもよい。このように、１つのウェルが複数の凹部又は凸部を有する場合、それぞれの凹部内又は凸部間で細胞塊を培養できるため、上記実施形態と同様に微細な細胞塊を容易に得ることができる。 The number of wells provided in the well plate may be one. Further, only one concave / convex concave portion or convex portion constituting the first pattern may be formed per well as in the above-described embodiment, or a plurality may be formed per well. As described above, when one well has a plurality of concave portions or convex portions, the cell mass can be cultured in each concave portion or between the convex portions, so that a fine cell mass can be easily obtained as in the above embodiment. it can.
　当該ウェルプレートの製造方法は、ウェルプレート本体や底面板に表面処理を施す工程をさらに備えてもよい。この表面処理としては、例えばウェルプレート本体や底面板の表面における濡れ性を向上させる処理等が挙げられる。また、この濡れ性向上処理としては、例えば紫外線照射、プラズマ処理等が挙げられる。ウェルプレート本体や底面板の表面における濡れ性を向上させることで、ウェル内周面や底面と培養液との親和性が向上し、ウェル内部に培養液を導入し易くなる。 The manufacturing method of the well plate may further include a step of performing a surface treatment on the well plate body or the bottom plate. Examples of the surface treatment include a treatment for improving wettability on the surface of the well plate main body or the bottom plate. Moreover, as this wettability improvement process, ultraviolet irradiation, a plasma process, etc. are mentioned, for example. By improving the wettability on the surface of the well plate main body and the bottom plate, the affinity between the inner peripheral surface and the bottom surface of the well and the culture solution is improved, and the culture solution is easily introduced into the well.
　当該ウェルプレート、ウェルプレート用シート及び培養方法は、三次元培養に限定されず、二次元培養等にも好適に使用できる。 The well plate, the well plate sheet and the culture method are not limited to three-dimensional culture, and can be suitably used for two-dimensional culture.
　以下、実施例に基づき本発明を詳述するが、この実施例の記載に基づいて本発明が限定的に解釈されるものではない。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not construed as being limited based on the description of the examples.
＜実施例１＞
　平均厚み１８８μｍの「ＺｅｏｎｏｒＦｉｌｍ、ＺＦ１４」（日本ゼオン製）を基材フィルムとして用い、ＳＣＩＶＡＸ社製の微細成形装置（ＦＬＡＮ２００）を使用し、表面に微細凹凸パターン（第２パターン）をその反転形状の金型を用いて成形した。第２パターンの形状は、凹部の平面視面積が９μｍ２の正方形で、各凹部が幅０．７μｍ、高さ１μｍの凸で囲まれた形状である。この成形は、まず基材フィルムを１７０℃に加熱し、金型をこの基材フィルム表面に押し付け、圧力４．０ＭＰａに達したところで６０秒間保持し、その後、金型の圧力を一定に保持しながら、１００℃以下まで冷却し、フィルムから離型することにより行った。 <Example 1>
Using a “ZeonorFilm, ZF14” (manufactured by ZEON Corporation) having an average thickness of 188 μm as a base film, a fine molding apparatus (FLAN200) made by SCIVAX is used, and a fine uneven pattern (second pattern) is formed on the surface of the inverted shape. Molded using a mold. The shape of the second pattern is a shape in which the recess has a square area in plan view of 9 μm 2 and each recess is surrounded by a protrusion having a width of 0.7 μm and a height of 1 μm. In this molding, the base film is first heated to 170 ° C., the mold is pressed against the surface of the base film, and is held for 60 seconds when the pressure reaches 4.0 MPa, and then the mold pressure is kept constant. However, it was performed by cooling to 100 ° C. or lower and releasing from the film.
　次に、上記基材フィルムを再度１７０℃に加熱し、直径０．７ｍｍ、深さ０．２ｍｍの半球状の金型を基材フィルム裏面に押し付け、圧力が４．０Ｍｐａに達したところで６０秒間保持した。その後、金型の圧力を一定に保持しながら、１００℃以下まで冷却し、フィルムから離型することで凹部（第１パターン）を形成した。 Next, the substrate film was heated again to 170 ° C., a hemispherical mold having a diameter of 0.7 mm and a depth of 0.2 mm was pressed against the back surface of the substrate film, and when the pressure reached 4.0 Mpa, 60 seconds. Retained. Thereafter, while keeping the pressure of the mold constant, the mold was cooled to 100 ° C. or less and released from the film to form a recess (first pattern).
　１のウェルにおける第１パターンの平面充填率は３０％であった。また、１のウェルにおける第２パターンの平面充填率は６６％であった。 The flat filling factor of the first pattern in one well was 30%. Further, the planar filling factor of the second pattern in one well was 66%.
　上記基材フィルムの表面をレーザー顕微鏡で観察したところ、転写領域全面に良好な微細凹凸パターンとこの微細凹凸パターンを表面に含む凹部構造が転写されていた。転写後の凹部の写真を図３に、微細凹凸パターンの写真を図４に示す。 When the surface of the base film was observed with a laser microscope, a good fine concavo-convex pattern and a concavo-convex structure including the fine concavo-convex pattern on the surface were transferred to the entire transfer region. The photograph of the recessed part after transcription | transfer is shown in FIG. 3, and the photograph of a fine uneven | corrugated pattern is shown in FIG.
　得られた平均厚さ１８８μｍのウェルプレート用シートを直径が６５ｍｍのシャーレ内に固定して細胞培養容器を作製した。 The obtained sheet for well plate having an average thickness of 188 μm was fixed in a petri dish having a diameter of 65 mm to prepare a cell culture container.
　作製した細胞培養容器内にＤ－ＭＥＭ培地（１０％牛胎児血清、１．２ｇ／ｌ炭酸水素ナトリウム、１００ＩＵ／ｍｌペニシリン、及び１００μｇ／ｍｌストレプトマイシンを含む）０．１ｍｌと共にヒト大腸がん由来の細胞株ＨＴ－２９細胞を４０００個播種した。これを３７℃、５％ＣＯ２の雰囲気下で５日間培養したところ、細胞塊が形成された。培地交換は、マイクロピペットを使用して培地を吸引除去し、新しい培地を入れるだけで容易に行うことができた。 In the prepared cell culture container, 0.1 ml of D-MEM medium (containing 10% fetal bovine serum, 1.2 g / l sodium bicarbonate, 100 IU / ml penicillin, and 100 μg / ml streptomycin) was derived from human colon cancer. 4000 cell lines HT-29 cells were seeded. When this was cultured at 37 ° C. in an atmosphere of 5% CO 2 for 5 days, a cell mass was formed. The medium was easily exchanged by simply removing the medium using a micropipette and adding a new medium.
　培養開始２日経過時にトミーデジタルバイオロジー社製の「Ｃｅｌｉｇｏ」（自動イメージング装置）にて細胞塊サイズの評価をしたところ、ＣＶ値（％）は６％であった。 When the cell mass size was evaluated with “Celigo” (automated imaging device) manufactured by Tommy Digital Biology after 2 days from the start of culture, the CV value (%) was 6%.
　培養開始５日経過時に再度細胞塊サイズの評価をしたところ、ＣＶ値は１５％であった。 When the cell mass size was evaluated again after 5 days from the start of culture, the CV value was 15%.
＜実施例２＞
　直径０．７ｍｍ、深さ０．２ｍｍの半球状の金型の代わりに直径０．３ｍｍ、深さ０．０５ｍｍの半球状の金型を使用した以外は実施例１と同様に細胞培養容器を作製した。 <Example 2>
A cell culture vessel was prepared in the same manner as in Example 1 except that a hemispherical mold having a diameter of 0.3 mm and a depth of 0.05 mm was used instead of the hemispherical mold having a diameter of 0.7 mm and a depth of 0.2 mm. Produced.
　１のウェルにおける第１パターンの平面充填率は１３％であった。 The flat filling factor of the first pattern in one well was 13%.
　作製した細胞培養容器を実施例１と同様に細胞塊サイズの評価をしたところ、２日間培養後のＣＶ値は７％、５日間培養後のＣＶ値は１０％であった。 When the produced cell culture container was evaluated for the cell mass size in the same manner as in Example 1, the CV value after 2 days of culture was 7%, and the CV value after 5 days of culture was 10%.
＜比較例１＞
　微細凹凸パターン（第２パターン）のみを有し、第１パターンを構成する凹凸の凹部及び凸部を有しないプレート（ＳＣＩＶＡＸライフサイエンス社の「ＮａｎｏＣｕｌｔｕｒｅ　Ｐｌａｔｅ　ＭＳパターン低接着」）を使用して実施例１と同様の手順で２日間培養後に細胞塊サイズの評価をしたところ、ＣＶ値は３０％であったため、それ以降の培養評価を行わなかった。 <Comparative Example 1>
Example using only concave and convex concave portions constituting the first pattern and a plate having no convex portions (“NanoCulture Plate MS pattern low adhesion” of SCIVAX Life Sciences) having only the fine concave and convex pattern (second pattern) When the cell mass size was evaluated after culturing for 2 days in the same procedure as in No. 1, the CV value was 30%.
＜比較例２＞
　微細凹凸パターン（第２パターン）を有せず、第１パターンを構成する凹凸の凹部のみを有するプレート（ＡＧＣテクノグラス社の「ＥＺ　ＳＰＨＥＲＥディッシュ３５ｍｍ」）を使用して実施例１と同様の手順で２日間培養後に細胞塊サイズの評価をしたところ、ＣＶ値は３４％であったため、それ以降の培養評価を行わなかった。 <Comparative Example 2>
The same procedure as in Example 1 using a plate (“EZ SPHERE dish 35 mm” manufactured by AGC Techno Glass Co., Ltd.) having only the concave and convex portions constituting the first pattern without having the fine concave and convex pattern (second pattern). When the cell mass size was evaluated after culturing for 2 days, the CV value was 34%. Therefore, the subsequent culture evaluation was not performed.
＜比較例３＞
　実施例１と同様の手順で基材フィルムに微細凹凸パターン（第２パターン）を形成せず、第１パターンを構成する凹凸の凹部のみを形成したウェルプレートを作製し、実施例１と同様に細胞培養容器を作製した。作製した細胞培養容器を実施例１と同様に細胞塊サイズの評価をしたところ、細胞培養液の交換が極めて困難であり、評価をすることができなかった。 <Comparative Example 3>
A well plate in which only the concave and convex portions constituting the first pattern were formed without forming the fine concave and convex pattern (second pattern) on the base film in the same procedure as in Example 1, and the same as in Example 1 was prepared. A cell culture vessel was prepared. When the cell culture vessel thus prepared was evaluated for the cell mass size in the same manner as in Example 1, it was extremely difficult to exchange the cell culture solution, and the evaluation could not be performed.
　以上説明したように、本発明のウェルプレートは製造が容易であり、かつ当該ウェルプレートによればサイズの均一性が高い細胞塊を容易に得ることができる。また、本発明のウェルプレート用シートによれば、容易に当該ウェルプレートを製造することができる。さらに、本発明の培養方法は、当該ウェルプレートを用いるため、サイズの均一性が高い細胞塊を容易に培養することができる。従って、本発明のウェルプレート、ウェルプレート用シート、及び培養方法は、今後さらに多用される細胞培養に好適に用いることができる。 As described above, the well plate of the present invention is easy to manufacture, and according to the well plate, a cell mass with high size uniformity can be easily obtained. Further, according to the well plate sheet of the present invention, the well plate can be easily manufactured. Furthermore, since the culture method of the present invention uses the well plate, it is possible to easily cultivate cell clusters with high size uniformity. Therefore, the well plate, well plate sheet, and culture method of the present invention can be suitably used for cell culture that will be used more frequently in the future.
１　ウェル
１ａ　第１パターン
１ｂ　第２パターン
２　底面
３　内周面
４　ウェルプレート本体
５　底面板
６　ウェルプレート用シート
７　貫通孔 DESCRIPTION OF SYMBOLS 1 Well 1a 1st pattern 1b 2nd pattern 2 Bottom surface 3 Inner peripheral surface 4 Well plate main body 5 Bottom plate 6 Well plate sheet 7 Through-hole

Claims (19)

  1. 　細胞を収容し、培養する１又は複数のウェルを備えるウェルプレートであって、
    　上記ウェルが底面に凹凸パターンを有し、
    　上記凹凸パターンが、
    　凹凸の組み合わせにより構成される第１パターンと、
    　上記第１パターンよりも微細な凹凸の組み合わせにより構成される第２パターンとの複合パターンであり、
    　上記第１パターンの表面に上記第２パターンが形成されていることを特徴とするウェルプレート。     A well plate comprising one or more wells for containing and culturing cells,
    The well has an uneven pattern on the bottom,
    The uneven pattern is
    A first pattern constituted by a combination of irregularities;
    It is a composite pattern with a second pattern constituted by a combination of fine irregularities than the first pattern,
    A well plate, wherein the second pattern is formed on a surface of the first pattern.
  2. 　上記第１パターンを構成する凹凸の凹部が上記ウェルプレートの底面に垂直な方向を軸とする立体形状である請求項１に記載のウェルプレート。 The well plate according to claim 1, wherein the concave and convex concave portions constituting the first pattern have a three-dimensional shape with a direction perpendicular to a bottom surface of the well plate as an axis.
  3. 　上記凹部が半球状である請求項２に記載のウェルプレート。 The well plate according to claim 2, wherein the concave portion is hemispherical.
  4. 　上記凹部が平底の有底筒状である請求項２に記載のウェルプレート。 The well plate according to claim 2, wherein the concave portion has a flat bottomed cylindrical shape.
  5. 　１のウェルにおける上記凹部の平均径が３０μｍ以上３０００μｍ以下である請求項１から請求項４のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 4, wherein an average diameter of the recesses in one well is 30 µm or more and 3000 µm or less.
  6. 　上記第１パターンが規則的パターンである請求項１から請求項５のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 5, wherein the first pattern is a regular pattern.
  7. 　上記第１パターンが平面視で単一形状の複数の凹部を有する請求項１から請求項６のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 6, wherein the first pattern has a plurality of concave portions having a single shape in plan view.
  8. 　１のウェルにおける上記凹部の平面充填率が１０％以上９９．９％以下である請求項１から請求項７のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 7, wherein a planar filling rate of the concave portion in one well is 10% or more and 99.9% or less.
  9. 　上記第２パターンを構成する凹凸の凹部が半球状である請求項１から請求項８のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 8, wherein the concave and convex portions constituting the second pattern are hemispherical.
  10. 　上記第２パターンを構成する凹凸の凹部が平底の有底筒状である請求項１から請求項８のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 8, wherein the concave and convex portions constituting the second pattern have a flat bottomed cylindrical shape.
  11. 　１のウェルにおける上記第２パターンを構成する凹凸の凹部の平面充填率が９９％以下である請求項１から請求項１０のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 10, wherein a planar filling rate of the concave and convex portions constituting the second pattern in one well is 99% or less.
  12. 　上記第２パターンを構成する凹凸の１の凹部の平面視面積が１００μｍ２以下である請求項１から請求項１１のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 11, wherein a planar view area of one concave portion of the concave and convex constituting the second pattern is 100 µm 2 or less.
  13. 　上記第２パターンが規則的パターンである請求項１から請求項１２のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 12, wherein the second pattern is a regular pattern.
  14. 　上記第２パターンが平面視で単一形状の複数の凹部を有する請求項１から請求項１３のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 13, wherein the second pattern has a plurality of concave portions having a single shape in plan view.
  15. 　細胞を収容し、培養する１又は複数のウェルを備えるウェルプレートのウェル底面を構成するシートであって、
    　基材フィルムを備え、
    　上記基材フィルムが一方の面のうち少なくとも上記ウェル領域に凹凸パターンを有し、
    　上記凹凸パターンが、
    　凹凸の組み合わせにより構成される第１パターンと、
    　上記第１パターンよりも微細な凹凸の組み合わせにより構成される第２パターンとの複合パターンであり、
    　上記第１パターンの表面に上記第２パターンが形成されていることを特徴とするウェルプレート用シート。     A sheet constituting a well bottom surface of a well plate including one or a plurality of wells for containing and culturing cells,
    A base film,
    The substrate film has an uneven pattern in at least the well region of one side,
    The uneven pattern is
    A first pattern constituted by a combination of irregularities;
    It is a composite pattern with a second pattern constituted by a combination of fine irregularities than the first pattern,
    A well plate sheet, wherein the second pattern is formed on a surface of the first pattern.
  16. 　細胞を収容し、培養する方法であって、
    　請求項１から請求項１４のいずれか１項に記載のウェルプレートを用い、
    　上記培養で得る細胞塊の平均径に応じて上記ウェルプレートの第１パターンの平面充填率を調整することを特徴とする培養方法。     A method for containing and culturing cells, comprising:
    Using the well plate according to any one of claims 1 to 14,
    A culture method comprising adjusting a plane filling rate of the first pattern of the well plate according to an average diameter of a cell mass obtained by the culture.
  17. 　上記培養が、三次元培養である請求項１から請求項１４のいずれか１項に記載のウェルプレート。 The well plate according to any one of claims 1 to 14, wherein the culture is a three-dimensional culture.
  18. 　上記培養が、三次元培養である請求項１５に記載のウェルプレート用シート。 The well plate sheet according to claim 15, wherein the culture is three-dimensional culture.
  19. 　上記培養が、三次元培養である請求項１６に記載の培養方法。 The culture method according to claim 16, wherein the culture is three-dimensional culture.
PCT/JP2017/020715 2016-06-03 2017-06-02 Well plate, well plate sheet, and culturing method WO2017209301A1 (en)

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WO2022210713A1 (en) * 2021-03-30 2022-10-06 国立大学法人北海道大学 Cell mass-forming member, culture container, method for producing cultured cells, and cultured cells with cell mass-forming member
WO2022266342A1 (en) * 2021-06-17 2022-12-22 Organos, Inc. Microwell array for high-throughput screening of micro-tissue and methods of using the same

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