JP6248476B2 - Excitation device - Google Patents

Description

  The present invention relates to a vibration device, for example, a vibration device used when vibrating a culture container for culturing cells such as a fertilized egg.

  Conventionally, fertilized eggs (zygotes) are produced by in vitro fertilization of sperm and ova in a culture system, not only in the livestock field but also in human infertility medicine. It is possible to culture from the zona pellucida through the zona pellucida to the stage of the escaped blastocyst, and the fertilized egg at the blastocyst stage from this cleavage is transferred to the uterus to obtain a litter Reproductive technology (ART) has been established.

  As a method for culturing fertilized eggs as described above, a culture solution of about 500 μL is placed in a well on a culture vessel, and a fertilized egg is cultured in the culture solution. A microdrop of about 20 μL is placed in a well on the culture vessel. A method is known in which the surface of the microdroplet is coated with mineral oil, and a fertilized egg is placed therein. In recent years, a method of individually managing a fertilized egg by providing a plurality of depressions on a culture container and introducing and cultivating a fertilized egg for each depression has been applied.

  By the way, when a plurality of depressions are provided on a culture container and a fertilized egg is introduced and cultured for each depression, it is known that bubbles may remain in the depression when the medium is introduced into the culture container. ing. In particular, when the culture vessel is formed of a highly hydrophobic material such as polystyrene or when a medium having a high surface tension is used, it has been confirmed by the present inventors that bubbles easily remain in the depression. ing. If air bubbles remain in the depression when introducing the culture medium into the culture vessel, there are concerns about changes in the culture environment, cell damage due to contact with the bubbles, contamination risk due to various bacteria, etc. Therefore, it is desired to develop a technique capable of reliably removing bubbles present in the depression.

  In response to such a request, Patent Document 1 discloses a method of shaking a plate, a method of drawing a vacuum from one surface of the plate, a method of using electrostatic force, a method of tilting a plate so that air can be replaced by buoyancy, and the like. Is disclosed. Patent Document 2 discloses a method using low-temperature plasma treatment, corona discharge treatment, ultraviolet irradiation, and the like, and a method of applying collagen, which is a protein that promotes cell adhesion.

JP 2009-96992 A JP 2007-228818 A

  According to the technique disclosed in Patent Document 1, air in a through hole that has been chemically treated so that the inner surface becomes hydrophilic can be replaced with liquid. However, in the technique disclosed in Patent Document 1, specific contents are not disclosed for each method, and it is possible to reliably remove bubbles remaining in a concave depression that is considered to be more difficult to remove bubbles. Whether or not is unknown.

  Further, according to the technique disclosed in Patent Document 2, although it is possible to prevent bubbles from adhering to the fine protrusions of the microwell, low temperature plasma treatment, corona discharge treatment, ultraviolet irradiation or the like is applied, or collagen is applied. Thus, it is necessary to form an organic film or an inorganic film in the cell culture container, which may cause a problem that the manufacturing cost of the cell culture container itself increases. Further, in the technique disclosed in Patent Document 2, the surface characteristics of a cell culture vessel subjected to low-temperature plasma treatment, corona discharge treatment, ultraviolet irradiation, or the like, or formed with an organic film or an inorganic film change due to secular change or the like. The problem that air bubbles easily remain in the microwell with the passage of time may also arise.

  The present invention has been made in view of the above-described problems, and the object thereof is, for example, without remaining a surface treatment or the like on a cell culture container, and bubbles remaining in a concave depression provided on the culture container It is an object of the present invention to provide a vibration apparatus that can easily and reliably remove the vibration.

  The vibration device of the present invention is obtained from an experiment by the present inventors that it is very easy to remove bubbles remaining in a concave depression by vibrating a culture container into which a culture medium has been introduced in a specific direction. Based on the findings.

  That is, the vibration device of the present invention is a vibration device that vibrates a culture container having a bottom surface and a side surface that define a culture region for culturing cells, and an installation unit for installing the culture container; And a vibration unit that vibrates the culture vessel installed in the installation unit in a direction orthogonal to the bottom surface.

  Moreover, the preferable form of an above-described vibration apparatus is that the said vibration part contacts the part of the said culture container installed in the said installation part, and vibrates the said culture container. . In a more preferable form of the above-described vibration device, the vibration part is embedded in the installation part, and at the time of vibration, a part of the vibration part extends from the installation part and contacts the lower surface of the culture vessel. Thus, the culture vessel is vibrated.

  Moreover, the preferable form of an above-described vibration apparatus is provided with the urging | biasing part which urges | biases the said culture container to the direction orthogonal to the said bottom face and the direction which goes to the said installation part in the said installation part. . In a further preferred form of the above-described vibration apparatus, the installation part has a defining part that defines the installation position of the culture vessel. In a further preferred form of the above-described vibration device, the culture container is installed at a position where the vibration unit and the culture container are in contact with each other at a position away from the center of the culture container.

  In a preferred embodiment of the above-described vibration device, the vibration unit is made of metal. In a further preferred form of the above-described vibration device, a contact portion of the vibration portion with the culture container is covered with an elastic member. Moreover, the further preferable form of an above-described vibration apparatus has the adjustment part in which the said vibration apparatus adjusts the output of the said vibration part.

  According to the vibration device of the present invention, a culture container in which a concave depression is formed on the bottom surface is used, for example, by exciting the culture container installed on the installation portion in a direction perpendicular to the bottom surface by the vibration unit. Thus, even when cells are cultured, bubbles remaining in the concave depression can be easily and reliably removed.

It is a figure which shows the basic composition of embodiment of the vibration apparatus which concerns on this invention, Comprising: (a) is the perspective view, (b) is the top view of the main-body part, (c) is the main-body part (D) is an enlarged cross-sectional view showing a part of the main body in an enlarged manner. It is a figure which shows an example of the culture container used with the vibration apparatus which concerns on this invention, Comprising: (a) is the perspective view, (b) expands and shows the cell accommodating part of the culture container of FIG. It is an enlarged plan view, (c) is a BB arrow view of FIG. 2b, (d) is the longitudinal cross-sectional view. It is a perspective view which shows an example of the lid | cover of the culture container shown in FIG. It is a flowchart explaining the method to culture | cultivate a cell in a culture container. 2 is a captured image obtained by capturing an image of a concave portion of a cell storage portion using a test sample of Example 1. FIG. It is the picked-up image which imaged the recessed part of the cell accommodating part by the test sample of Example 2. FIG. It is a figure which shows the relationship between an excitation frequency and a bubble removal rate.

  Hereinafter, embodiments of a vibration device according to the present invention will be described with reference to the drawings.

  FIG. 1 is a diagram showing a basic configuration of an embodiment of a vibration device according to the present invention, FIG. 1a is a perspective view thereof, and FIG. 1b is an upper surface of a main body portion of the vibration device shown in FIG. 1a. 1c is a side view of the main body of the vibration device shown in FIG. 1a, and FIG. 1d is an enlarged cross-sectional view showing a part of the main body of the vibration device shown in FIG. 1a. is there.

  As shown in FIG. 1a, the vibration device 50 of the present embodiment mainly includes an installation unit 20 for installing the culture vessel 10 and a culture vessel 10 installed in the installation unit 20 in the vertical direction (the culture vessel 10). A main body 30 having a vibration portion 21 that vibrates in the direction perpendicular to the upper surface (bottom surface) 1a (see FIG. 2a) of the bottom portion 1 and the output (amplitude) of the vibration portion 21 of the main body portion 30. An adjustment unit 40 for adjustment is provided, and the main body unit 30 and the adjustment unit 40 are electrically connected via a communication cable 39. The main body 30 and the adjustment unit 40 may be configured integrally without using the communication cable 39.

  The installation portion 20 of the main body 30 includes a substantially cylindrical casing 20a, an upper plate 20b provided at the upper end of the casing 20a, and a bottom plate 20c provided at the lower end of the casing 20a. An opening 20e is formed (see FIG. 1b). The vibration unit 21 of the main body 30 is made of, for example, a metal vibrator so as to maintain rigidity. The vibration unit 21 is embedded in the housing 20a of the installation unit 20, and the tip (contact portion with the culture vessel 10) 21a is located on the upper side. It is slidably disposed in the opening 20e of the plate 20b so that the tip 21a of the vibrating unit 21 extends from the upper surface 20d of the upper plate 20b and contacts the lower surface of the bottom 1 of the culture vessel 10 during vibration. It has become.

  In addition, in order to relieve the impact to the bottom part 1 of the culture container 10 at the time of vibration, the front-end | tip part 21a of the vibration part 21 is covered with the elastic member 21b, for example (refer FIG. 1d). The material of the elastic member 21b is not particularly limited, and specific examples include nitrile rubber, silicone rubber, urethane rubber, acrylic rubber, butadiene rubber, styrene butadiene rubber, fluorine rubber, and ethylene propylene rubber in addition to natural rubber. It is done. Further, the shape of the upper surface of the distal end portion 21a of the vibrating portion 21, particularly the upper surface of the elastic member 21b provided on the distal end portion 21a of the vibrating portion 21 (contact surface with the culture vessel 10) is not particularly limited. It may be a shape, or a shape that is convex upward.

  On the upper plate 20b of the installation portion 20, a rod-like support portion 20f is erected, and an urging portion 20g made of a leaf spring or the like is attached to the support portion 20f. The culture vessel 10 installed on the upper plate 20b of the installation unit 20 is biased in the direction of the upper plate 20b (vertically downward) by the urging unit 20g, and the culture vessel 10 is placed on the installation unit 20. It is sandwiched between the plate 20b and the urging portion 20g (see FIG. 1c).

  The upper plate 20b of the installation unit 20 is provided with a defining unit (not shown) that defines the position where the culture vessel 10 is installed. The defining portion may be a mark that visually defines the installation position of the culture vessel 10, or a protrusion that physically defines the installation location of the culture vessel 10. The culture vessel 10 installed on the upper plate 20b of the installation unit 20 by this defining unit is located at a position where the tip 21a of the vibration unit 21 and the bottom 1 of the culture vessel 10 are separated from the center of the culture vessel 10, in particular. The tip 21a of the vibration unit 21 and the bottom 1 of the culture vessel 10 are installed so as to contact at a position other than the cell storage unit 3 of the culture vessel 1 described later.

  In addition, an adjustment dial 40a for setting the output (amplitude) of the excitation unit 21 to a predetermined output is disposed on the front surface of the adjustment unit 40.

  Here, an example of the culture vessel 10 used in the vibration device 50 of the present embodiment will be outlined. The cells cultured in the culture vessel 10 are, for example, cells that need to be individually managed, and need to specify individual cells during and after culture, that is, a plurality of cells are cultured. Cells that are undesirable to be confused with each other in the culture vessel. Examples of cells that require individual management include fertilized eggs, egg cells, ES cells (embryonic stem cells), iPS cells (artificial pluripotent stem cells), and the like. An egg cell refers to an unfertilized egg cell, and includes an immature oocyte and a mature oocyte. After fertilization, the fertilized egg increases in number of cells from the 2 cell stage, the 4 cell stage, and the 8 cell stage by cleavage, and develops into a blastocyst through a morula. Fertilized eggs include early embryos such as 2-cell embryos, 4-cell embryos and 8-cell embryos, morulas, blastocysts (including early blastocysts, expanded blastocysts and escaped blastocysts). A blastocyst means an embryo composed of external cells with the potential to form the placenta and internal cell masses with the potential to form embryos. ES cells refer to undifferentiated pluripotent or totipotent cells obtained from the inner cell mass of a blastocyst. An iPS cell refers to a cell having a pluripotency similar to that of an ES cell by introducing several types of genes (transcription factors) into somatic cells (mainly fibroblasts). That is, the cells to be cultured in the present invention include a collection of a plurality of cells such as fertilized eggs and blastocysts. The vibratory apparatus of the present invention is preferably suitable for culturing mammalian and avian cells, particularly mammalian cells. Mammals refer to warm-blooded vertebrates, for example, primates such as humans and monkeys, rodents such as mice, rats and rabbits, pets such as dogs and cats, and domestic animals such as cattle, horses and pigs. Etc.

  As shown in FIG. 2 a, the culture vessel 10 has a substantially disk-shaped bottom 1 and a side wall 2 standing from the outer edge of the bottom 1, and an upper surface (bottom surface) 1 a of the bottom 1 and the inner periphery of the side wall 2. A culture region for culturing cells is defined from the surface (side surface) 2a. A cell accommodating portion 3 having a recess (also referred to as a microwell) 4 for accommodating cells is provided at substantially the center of the bottom portion 1, and an inner wall 5 is erected around the cell accommodating portion 3. In addition, as shown in FIG. 2 b, 25 concave portions 4 of the cell containing portion 3 are provided close to each other in a 5 × 5 square lattice shape along the upper surface 1 a of the bottom portion 1. As shown in FIG. 2c, the opening 4 is formed of a side surface 4a substantially perpendicular to the upper surface 1a of the bottom 1 and a bottom surface 4b substantially parallel to the upper surface 1a. Further, as shown in FIG. 2d, the inner wall 5 is formed with a height lower than that of the side wall 2, and a medium for introducing the medium from the cell accommodating portion 3 provided in the bottom 1 and the inner peripheral surface 5a of the inner wall 5 is provided. An area is defined.

  In addition, the shape in particular of the bottom part 1 which comprises the culture container 10 is not restrict | limited, Polygon shapes, such as a triangle and a quadrangle | tetragon, and elliptical shape may be sufficient. In addition, the concave portion 4 of the cell storage portion 3 may be formed as a depression directly on the surface of the bottom portion 1 of the culture vessel 10, or the cell storage portion 3 is formed by forming the cell storage portion 3 with a member protruding from the bottom portion 1. You may form as a hollow in the surface of. Moreover, the cell container 3 may be provided with a plurality of cell accommodating portions 3, and the number and arrangement of the recesses 4 of the cell accommodating portion 3 and the shapes and dimensions of the openings, the side surfaces 4 a and the bottom surface 4 b of each recess 4 are accommodated. You may change suitably according to the kind of cell to do.

  For example, when fertilized eggs are cultured, it is desirable to culture to the blastocyst stage, so the diameter (opening width) of the circular opening of the recess 4 is larger than the maximum dimension of the cells in the blastocyst stage. It is desirable. Since the maximum size of cells in the blastocyst stage is usually 100 μm to 280 μm, the diameter of the circular opening is usually 100 μm or more. Further, for example, when the side surface 4a of the recess 4 is inclined in a conical shape or a truncated cone shape so that its height increases from the lowest position of the recess 4 toward the outer edge of the recess 4, It becomes easy to move to the place (the deepest part of the recessed part 4) to arrange. Further, for example, in order to hold the cell in the recess 4, the depth of the recess 4 is preferably not less than 1/3 of the maximum diameter of the cell, and more preferably not less than 1/2. Further, for example, in order to facilitate the introduction of the culture medium into the recess 4, the depth of the recess 4 is preferably not more than 1 times the recess diameter, and particularly preferably not more than 1/2. Moreover, since the convection is less likely to occur as the diameter of the recess 4 is smaller and the depth thereof is deeper, there is a possibility that a change in the composition of the surrounding culture solution is likely to occur with cell respiration and metabolism. Since the ease of growth of cells changes under the influence of the composition of the surrounding culture medium, it is preferable to set the diameter and depth in consideration of biological effects so as to promote cell growth. In general, the depth of the recess 4 is preferably about 50 μm to 200 μm, although it depends on the type of cell.

  In addition, the material of the culture vessel 10 is not particularly limited, and specifically, inorganic materials such as metal, glass and silicon, plastic (for example, polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, ABS resin, nylon, An acrylic resin, a fluororesin, a polycarbonate resin, a polyurethane resin, a methylpentene resin, a phenol resin, a melamine resin, an epoxy resin, and a vinyl chloride resin). The culture vessel 10 can be manufactured by a method known to those skilled in the art. For example, when a culture container made of a plastic material is manufactured, it can be manufactured by a conventional molding method such as injection molding. Furthermore, the culture vessel 10 may be subjected to a surface treatment or a surface coat that promotes the development of a fertilized egg. In particular, in order to promote the development of a fertilized egg, when co-culturing with cells of other organs (for example, endometrial cells or fallopian tube epithelial cells), these cells need to be adhered to a culture vessel in advance. . In such a case, it is advantageous to coat the surface of the culture vessel with a cell adhesive material.

  The culture vessel 10 can have a lid 11 shown in FIG. 3, for example, as needed. The culture vessel 10 is vibrated by the vibration device 50 in a posture with the lid 11 attached, for example.

  Next, an outline of a method for filling the culture vessel 10 with a medium using the above-described vibration device 50 and culturing cells in the culture vessel 10 will be described. As shown in FIG. 4, this method mainly comprises a preparation step (S11), a medium introduction step (S12), an oil introduction step (S13), a vibration step (S14), and a heat retention step (S15). ing.

[Preparation process]
First, for culturing cells, for example, the culture vessel 10 shown in FIG. 2 is prepared (S11).

[Medium introduction process]
Next, a medium containing a fertilized egg or the like using a pipette, a glass capillary, or the like in a medium region defined in the cell container 3 of the culture vessel 10, particularly the inner wall 5 standing around the cell container 3. Is introduced (S12). The medium is introduced up to the height of the inner wall 5 and is held so as not to leak out of the inner wall 5 due to the surface tension of the medium. The culture medium is not particularly limited as long as it has the ability to culture cells. Examples of the culture medium for fertilized egg culture include M16 and CR1aa.

[Oil introduction process]
Next, mineral oil is introduced into the culture region defined by the inside of the side wall 2 of the culture vessel 10, that is, the upper surface 1a of the bottom 1 of the culture vessel 10 and the inner peripheral surface 2a of the side wall 2 (S13). Mineral oil is introduced to a height that covers the medium introduced into the culture vessel 10, thereby preventing the medium from drying.

[Excitation process]
As described above, since the concave portion 4 of the cell accommodating portion 3 is extremely small, bubbles may remain in the concave portion 4 of the cell accommodating portion 3 during the culture medium introducing step of S12. In particular, when the culture vessel 10 is formed of a highly hydrophobic material such as polystyrene, or when a medium with high surface tension is used, the possibility increases. Therefore, for example, after the lid 11 is attached to the culture container 10 in which the medium and mineral oil are stored, the culture container 10 is vibrated by the vibration device 50 to remove bubbles present in the recess 4 of the cell storage unit 3. (S14).

  Specifically, the culture vessel 10 is installed at a predetermined position on the upper plate 20b by the defining portion provided on the upper plate 20b of the installation unit 20 of the vibration device 50, and the upper side of the lid 11 of the culture vessel 10 is placed. The urging portion 20g attached to the support portion 20f is disposed in contact with the urging portion 20g, and the culture vessel 10 is urged toward the upper plate 20b (vertically downward) by the urging portion 20g. The culture vessel 10 is sandwiched and fixed by the plate 20b and the urging portion 20g (see FIG. 1c). In this state, the vibration unit 21 is driven, and the tip 21a of the vibration unit 21 is the lower surface of the bottom 1 of the culture vessel 10 installed in the installation unit 20, particularly the lower surface of the culture vessel 1 at a position other than the cell storage unit 3. The culture vessel 10 is vibrated in a direction orthogonal to the upper surface (bottom surface) 1a of the bottom 1 (see FIG. 1d). At that time, the portion of the urging portion 20g that contacts the lid 11 of the culture vessel 10 also vibrates in a direction (vertically downward) perpendicular to the upper surface (bottom surface) 1a of the bottom 1 of the culture vessel 10.

  The top surface of the tip portion 21a of the vibration portion 21, particularly the top surface of the elastic member 21b attached to the tip portion 21a of the vibration portion 21, is the same as the top surface of the upper plate 20b of the installation portion 20 at the start of vibration. It may be set to one, or may be set slightly protruding from the upper surface of the upper plate 20b. Further, the supporting portion 20f and the urging portion 20g attached to the supporting portion 20f are omitted, and the user can hold the culture vessel 10 on the upper plate 20b of the installation portion 20 when the culture vessel 10 is vibrated. Good. When the culture vessel 10 is vibrated, the culture vessel 10 vibrates from the outer peripheral surface of the side wall 2 of the culture vessel 10 or the upper surface of the lid 11 of the culture vessel 10 in addition to the lower surface of the bottom 1 of the culture vessel 10. May be given. Furthermore, the position at which the tip 21a of the vibration unit 21 and the bottom 1 of the culture vessel 10 are in contact with each other is not particularly limited, and the tip 21a of the vibration unit 21 and the culture vessel 10 are approximately at the center of the bottom 1 of the culture vessel 10. The bottom 1 may be brought into contact.

  The amplitude or the like of the culture vessel 10 in this vibration step S14 depends on the shape and size of the opening of each recess 4 and the side surface 4a and the bottom surface 4b, the natural frequency of the culture vessel 10 itself, the type of cells to be accommodated, etc. Adjustment can be performed using an adjustment dial 40 a provided on the front surface of the adjustment unit 40. However, since it is advantageous to introduce a larger amount of medium into the medium region inside the inner wall 5 erected around the cell accommodating portion 3, the excitation frequency and amplitude of the culture vessel 10 are different from those of the inner wall 5. It is desirable that the medium introduced up to the height is set to a value that does not leak to the outside of the inner wall 5 due to the surface tension of the medium, and that the bubbles in the recesses 4 of the cell accommodating part 3 can be detached from the recesses 4. . The excitation frequency of such a culture vessel 10 is, for example, about 5 kHz to 1 MHz as will be described based on the following examples. Further, the amplitude in the vertical direction is about 50 μm or more equivalent to the depth of the recess 4 since the depth of the recess 4 is about 50 μm to 200 μm as described above, and considering the surface tension of the culture medium, for example, It is preferable that it is about 1 mm or less. Further, since the bubble size in the recess 4 is about 50 μm, the vertical amplitude is sufficient if the bubble can be slid in the recess 4, for example, about 1/10 or more of the bubble size, that is, about 5 μm or more. There may be.

[Heat retention process]
Then, as described above, the culture container 10 is vibrated using the vibration device 50 to remove bubbles present in the concave portion 4 of the cell container 3, and then the culture container 10 is necessary for the growth and maintenance of the cultured cells. Is introduced into an incubator that provides an environmental atmosphere containing a certain gas and a constant environmental temperature (S15). Necessary gases include water vapor, free oxygen (O ₂ ) and carbon dioxide (CO ₂ ). By adjusting the environmental temperature and the CO ₂ content, the pH of the medium can be stabilized within a certain time. A stable pH is obtained by a stable CO ₂ content and a stable temperature. Note that the culture conditions such as temperature, gas, and culture medium can be adjusted by, for example, comparing an image of a cell in culture with an image that has been captured and stored in advance using an image comparison program. . For example, the culture conditions of the incubator when cultivating rat fertilized eggs are 5% CO ₂ , 5% O ₂ , 90% air, 37 ° C., and humidity saturation.

  Thus, according to the vibration device 50 of the present embodiment, the culture vessel 10 into which the culture medium has been introduced is in the vertical direction, that is, in the direction perpendicular to the upper surface (bottom surface) 1a of the bottom 1 of the culture vessel 10 and the recess 4 For example, even when cells are cultured using the culture vessel 10 in which the concave portion 4 is formed on the upper surface (bottom surface) 1a of the bottom portion 1 by being vibrated toward the opening direction of the bottom portion 1, it exists in the concave portion 10. Air bubbles can be easily and reliably removed. In addition, by introducing the culture vessel 10 after being shaken by the shaker 50 into an incubator that provides a constant environmental temperature, the bubbles remaining in the recess 4 after being shaken by the shaker 50 are expanded or activated. Since it can be made to detach | leave from the said recessed part 4, the bubble which exists in the recessed part 4 of the cell accommodating part 3 can be removed more reliably.

  In the method described above, the vibration step was performed after the oil introduction step, but the vibration step using the vibration device 50 may be performed after the medium introduction step or introduced into the culture vessel 10. If the medium can be allowed to evaporate, the oil introduction step itself may be omitted. When the culture container is vibrated before the mineral oil is introduced into the culture region, the culture medium is easily leaked to the outside of the inner wall 5, and accordingly, the culture container 10 is adjusted using the adjustment dial 40a of the adjustment unit 40 or the like. It is necessary to adjust the excitation frequency, amplitude, and the like.

<Experiment and result of measuring bubble removal rate of recesses by test sample of example>
The present inventors have taken images of the recesses of the cell storage portion in a plurality of culture vessels (Examples 1 and 2 and Comparative Example 1) that are vibrated at different vibration frequencies using the vibration device of the above-described form. And the bubble removal rate of a recessed part was measured from the captured image.

  An outline of a method for measuring the bubble removal rate is as follows. A culture vessel 10 having the form shown in FIG. The culture vessel was made of polystyrene resin, and the bottom portion had a thickness of about 1 mm, the inner wall had a thickness of about 0.5 mm, the inner wall had an inner circumference of about φ7 mm, and the inner wall had a height of about 2 mm. Moreover, the diameter of the opening part of the recessed part of a cell accommodating part was about 280 micrometers, the depth of the recessed part was about 160 micrometers, and the space | interval between the centers of adjacent recessed parts was about 400 micrometers. After introducing 70 μL of CR1aa into the culture region of the culture vessel and introducing mineral oil into the culture region of the culture vessel, an image of the concave portion of the cell storage portion was taken with an imaging camera (this captured image is referred to as image 1). . And the culture container is installed in the vibration apparatus of the form shown in FIG. 1, and it is 1 MHz (Example 1), 40 kHz (Example 2), 1 kHz toward the orthogonal | vertical direction, ie, the direction orthogonal to the upper surface of the bottom part of a culture container. After shaking in (Comparative Example 1), an image of the concave portion of the cell storage portion is taken by an imaging camera (this captured image is referred to as image 2), and both the captured images 1 and 2 are compared, and the following formula 1, the removal rate of bubbles in the recess was calculated.

(Equation 1)
1- (number of recesses where bubbles exist in image 2) / (number of recesses where bubbles exist in image 1)

  In the test sample of Example 1, as shown in FIG. 5, the bubble removal rate in the recess was about 32% (1-17 / 25). Moreover, in the test sample of Example 2, as shown in FIG. 6, the bubble removal rate in the recess was about 100%. Further, in the test sample of Comparative Example 1, the bubbles in the recesses were hardly removed.

  From this experimental result, by using the vibration device, the culture container is vibrated in the vertical direction, that is, in the direction orthogonal to the top surface of the bottom part and in the direction in which the concave part is opened. It has been demonstrated that bubbles present in the recess can be removed. In particular, as shown in FIG. 7, if the excitation frequency at the time of excitation of the vibration apparatus is 5 kHz or more and less than 1 MHz, about 1/3 or more of the bubbles present in the recesses of the cell storage part can be removed. Was confirmed.

1: bottom portion, 1a: top surface (bottom surface) of bottom portion, 2: side wall, 2: inner peripheral surface (side surface) of side wall, 3: cell accommodating portion, 4: concave portion of cell accommodating portion, 4a: side surface of concave portion, 4b: Bottom of recess, 5: inner wall, 10: culture vessel, 11: lid, 20: installation part, 20e: opening, 20g: biasing part, 21: vibration part, 21a: tip part of vibration part, 21b: Elastic member, 30: body part, 39: communication cable, 40: adjustment part, 40a: adjustment dial, 50: vibration device

Claims (8)

A vibration apparatus that vibrates a culture container that has a bottom surface and a side surface that define a culture region for culturing cells, and is provided with a cell container having a recess for accommodating the cells on the bottom surface. Because
An installation part for installing the culture vessel;
An excitation unit that vibrates the culture vessel in a direction perpendicular to the bottom surface in contact with the lower surface of the culture vessel installed in the installation unit;
An exciting device comprising: a defining part that defines an installation position of the culture container so that the vibration part and the culture container are in contact with a lower surface of the culture container at a position other than the cell storage part.   The vibration device according to claim 1, wherein an inner wall is erected around the cell storage portion.   The vibration unit is embedded in the installation unit, and at the time of vibration, a part of the vibration unit extends from the installation unit and contacts the lower surface of the culture vessel to vibrate the culture vessel The vibration exciter according to claim 1 or 2.   The urging unit according to any one of claims 1 to 3, wherein the installation unit is provided with an urging unit that urges the culture vessel in a direction orthogonal to the bottom surface and in a direction toward the installation unit. Shaker.   The vibration apparatus according to any one of claims 1 to 4, wherein the culture container is installed at a position where the vibration unit and the culture container are in contact with each other at a position away from the center of the culture container. .   The excitation device according to any one of claims 1 to 5, wherein the excitation unit is made of metal.   The excitation device according to any one of claims 1 to 6, wherein a contact portion of the excitation unit with the culture vessel is covered with an elastic member.   The said vibration apparatus is an vibration apparatus as described in any one of Claim 1 to 7 which has an adjustment part which adjusts the output of the said vibration part.

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