Culture device, method for observing object to be cultured, and culture chip

Abstract

This culture device is provided with a container main body for accommodating a culture solution, and a culture chip for forming a culture chamber in the interior of the container main body, the container main body being provided with a bottom wall part having light transmittance and air permeability, the culture chip being provided with a frame part and a film member, the frame part being in contact with the upper surface of the bottom wall part, and the film member being in contact with the upper surface of the bottom wall part. The membrane member has liquid permeability or liquid-containing substance permeability, is fixed to the upper surface of the frame part, and forms the culture chamber with the bottom wall part and the frame part.

Description

Technical Field

[0001] The present invention relates to a culture device, a culture object observation method and a culture chip.

[0002] This application claims priority based on Japanese Patent Application No. 2022-177449 ​​filed in Japan on November 4, 2022, and the contents are incorporated herein by reference. Background Art

[0003] Patent Document 1 listed below discloses a culture medium additive containing agar having a weight average molecular weight of 10,000 to 60,000, and a method for culturing cells or tissues in a state where cells or tissues are dispersed in the culture medium composition.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application Publication No. 2022-25134 Summary of the invention

[0007] Problems to be solved by the invention

[0008] In recent years, the serious decline in birthrates has intensified, and the number of infertility patients has increased. About half of infertility patients are male infertility. Although about 90% of male infertility is caused by sperm production disorders, due to the complexity of sperm production phenomena, there are many unknown details of the sperm production mechanism. This is also evidenced by the fact that there is currently almost no effective treatment for male infertility.

[0009] Spermatozoa are formed by cell division, differentiation, and meiosis in spermatogenic tubules with a diameter of less than 200 μm located in the testis. In the field of infertility treatment or embryology, efforts are made to clarify the mechanism of spermatogenesis by observing the different morphological changes from spermatogonia to sperm in time and space.

[0010] As a method for observing the whole picture of the spermatogenesis process, an in vitro (in vivo) experimental system as described in the above-mentioned Patent Document 1 has been proposed. As this in vitro experimental system, for example, there is a method (conventional organ culture method) of culturing testicular tissue taken out from mice on an agarose gel block (agar) immersed in a culture medium. According to this method, long-term culture of several months can be carried out, and spermatogenesis in vitro can be achieved.

[0011] When observing tissues in the above-mentioned conventional organ culture method, the state of the tissues is simply observed using a stereo microscope. On the other hand, in order to observe the production process of sperm smaller than 10 μm in detail, it is necessary to use an inverted microscope and a high-magnification lens for bright field-fluorescence observation.

[0012] Yet, in above-mentioned organ culture method in the past, the thickness and color of agarose gel block can hinder optical observation, and therefore it is difficult to utilize inverted microscope to carry out high-precision observation while cultivating.Therefore the current situation is: when needs detailed observation, testis tissue has to be moved to the sheet material used for observation and implement bright field-fluorescence observation based on inverted microscope.

[0013] However, this operation not only causes damage to testicular tissue or causes contamination, but also has a fatal disadvantage that the temporal resolution of observation cannot be improved.

[0014] An object of the present invention is to provide a culture device, a culture object observation method, and a culture chip that solve the above-mentioned problems.

[0015] Means for solving problems

[0016] A culture device according to one embodiment of the present invention comprises a container body for containing a culture solution, and a culture chip forming a culture chamber inside the container body, the container body comprising a bottom wall portion having light transmittance and air permeability, the culture chip comprising a frame portion and a membrane member, the frame portion abutting against an upper surface of the bottom wall portion, the membrane member being liquid permeable or liquid-containing material permeable and fixed to an upper surface of the frame portion, forming the culture chamber together with the bottom wall portion and the frame portion.

[0017] In the above-mentioned culture apparatus, oxygen in a gas phase may be supplied to the culture object accommodated in the above-mentioned culture chamber via the above-mentioned bottom wall portion, and the above-mentioned culture solution may be supplied via the above-mentioned membrane member.

[0018] In the above-mentioned culture apparatus, the thickness of the culture object accommodated in the above-mentioned culture chamber may be controlled by the height of the above-mentioned frame portion.

[0019] In the above culture device, the membrane member may be light-transmissive.

[0020] In the above culture device, the frame may be fixed to the upper surface of the bottom wall. In the above culture device, the frame may have a height in a range of 20 μm to 400 μm.

[0021] In the above-mentioned culture device, the culture object may be a seminiferous tubule, and the frame may have a height within a range of 50 μm to 350 μm.

[0022] In the above culture device, the culture object may be a mouse testis tissue, and the frame may have a height within a range of 50 μm to 200 μm.

[0023] In the above culture device, the upper surface of the bottom wall portion may be hydrophobic.

[0024] In the above culture apparatus, a portion of the upper surface of the bottom wall portion that forms the culture chamber may be hydrophobic, and a portion other than the portion that forms the culture chamber may be hydrophilic.

[0025] In the culture object observation method according to one aspect of the present invention, the culture device is placed on an inverted microscope, and the culture chamber is observed through the bottom wall portion.

[0026] A culture chip according to one embodiment of the present invention comprises: a membrane member having light-transmitting properties and liquid-permeating properties; and a frame portion fixed to one surface of the membrane member.

[0027] In the above-mentioned culture chip, the membrane member may be light-transmissive.

[0028] A cell culture method according to one embodiment of the present invention is a cell culture method using the culture apparatus, wherein oxygen in a gas phase is supplied to a culture object accommodated in the culture chamber via the bottom wall portion, and the culture solution is supplied via the membrane member.

[0029] Effects of the Invention

[0030] According to one embodiment of the present invention, the temporal resolution of observation can be improved without damaging or contaminating the culture object. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] [ Figure 1 ] is a structural diagram of the culture device of the first embodiment.

[0032] [ Figure 2 ] is a bottom view of the culture chip of the first embodiment.

[0033] [ Figure 3 ] is an explanatory diagram of the culture object observation method of the first embodiment.

[0034] [ Figure 4 ] is a diagram showing the structure of a culture device of a comparative example.

[0035] [ Figure 5 ] is a diagram showing the results of a testicular tissue culture experiment using a culture device of a comparative example.

[0036] [ Figure 6 ] is a diagram showing the results of a testis tissue culture experiment using the culture device of the first embodiment.

[0037] [ Figure 7 ] is a graph showing the postnatal age and seminiferous tubule growth rate of the testicular tissue of the first embodiment and the comparative example.

[0038] [ Figure 8 ] is a table summarizing the relationship between pore size and visibility of culture objects when polycarbonate is used as a membrane component.

[0039] [ Fig. 9 ] is a comparison photograph of the bright field and the fluorescent field (RFP) using an inverted microscope when polycarbonate with a pore size of 0.4 μm is used as a membrane component.

[0040] [ Fig.10 ] is a comparison photograph of the bright field and the fluorescent field (RFP) using an inverted microscope when polycarbonate with a pore size of 10 μm is used as a membrane component.

[0041] [ Fig.11 ] is a table summarizing the relationship between pore size and visibility of culture objects when polyethylene terephthalate is used as a membrane component.

[0042] [ Fig.12 ] is a comparison photograph of the bright field and the fluorescence field (RFP) using an inverted microscope when polyethylene terephthalate with a pore size of 0.45 μm is used as a membrane member.

[0043] [ Fig.13 ] is a comparison photograph of the bright field and the fluorescent field (RFP) using an inverted microscope 30 when polyethylene terephthalate with a pore size of 3 μm is used as a membrane member.

[0044] [ Fig.14 ] is a table summarizing the materials, pore sizes, porosity, and light transmittance of the membrane components used in the first embodiment.

[0045] [ Fig.15 ] is a graph showing the postnatal age and Acr-GFP expression rate of the testicular tissue of the second example and the comparative example.

[0046] [ Fig.16 ] is a figure comparing the fluorescence image of the testicular tissue at postnatal age (35th day) when the comparative example is used with polydimethylsiloxane (PDMS) as the bottom wall part.

[0047] [ Fig.17 ] is a figure comparing fluorescent images of testicular tissue at postnatal age (35th day) when TPX (registered trademark), a 4-methyl-1-pentene polymer, and Lumox (registered trademark), a tetrafluoroethylene-hexafluoropropylene copolymer (FEP), were used as the bottom wall.

[0048] [ Fig.18 ] is a diagram showing a situation in which a culture solution and a culture object are set in a culture device according to a third embodiment.

[0049] [ Fig.19 ] is a diagram showing a situation in which a culture solution and a culture object are set in a culture device in which the upper surface of the bottom wall is made hydrophilic.

[0050] [ Fig. 20 ] is a diagram showing a situation in which a culture solution and a culture object are set in a culture device of a variation example of the third embodiment.

[0051] [ Fig.21 ]yes Fig. 20 A top view of the container body is shown. DETAILED DESCRIPTION

[0052] Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[0053] (First Embodiment)

[0054] Figure 1 It is a diagram showing the configuration of the culture apparatus 1 according to the first embodiment.

[0055] like Figure 1 As shown, the culture device 1 includes a container body 10 and a culture chip 20 .

[0056] The container body 10 has a culture solution storage chamber 10A formed therein for storing a culture solution 2. The culture chip 20 is disposed in the culture solution storage chamber 10A, and forms a culture chamber 4 for culturing a culture object 3. In the present embodiment, a testis tissue is exemplified as the culture object 3. It should be noted that the culture object 3 may be, for example, other organ tissues such as ovarian tissue, liver tissue, kidney tissue, pancreatic tissue, lung tissue, intestinal tissue, etc., or may be a tissue slice (a material obtained by cutting a tissue), a cell mass (organoid, spheroid, etc.), a cell, a bacterium, etc.

[0057] The container body 10 is formed into a bottomed cylindrical shape. Figure 1 The container body 10 shown is a single container, but it can also be a structure in which a plurality of container bodies 10 are connected like a multi-well plate. The container body 10 has a side wall portion 11 and a bottom wall portion 12. The side wall portion 11 is formed in a cylindrical shape surrounding the periphery of the culture liquid storage chamber 10A. The side wall portion 11 can be cylindrical or square. The opening on the upper surface 11a side of the side wall portion 11 is open, and the opening on the lower surface 11b side of the side wall portion 11 is closed by the bottom wall portion 12.

[0058] The bottom wall 12 has light transmittance and air permeability. The material of the bottom wall 12 is polydimethylsiloxane (PDMS), which is a kind of silicone. In addition, as long as the bottom wall 12 has light transmittance and air permeability, its material is not limited to polydimethylsiloxane. For example, as the material of the bottom wall 12, 4-methyl-1-pentene polymers and tetrafluoroethylene-hexafluoropropylene copolymers (FEP) can also be used. As the light transmittance of the bottom wall 12, it is preferably colorless and transparent, and as the air permeability of the bottom wall 12, it is sufficient as long as at least oxygen and carbon dioxide can pass through. Specifically, the oxygen permeability of the bottom wall 12 at a temperature of 23°C and a humidity of 0% is 4500 to 90000cm 3 / (m 2 ×24h×atm), preferably 4500~67500cm 3 / (m 2 ×24h×atm), more preferably 4500~47000cm 3 / (m 2 ×24h×atm), more preferably 4500 to 45000cm 3 / (m 2 ×24h×atm). The upper surface 12a of the bottom wall portion 12 is fixed to the lower surface 11b of the side wall portion 11 by bonding or the like.

[0059] The material of the side wall 11 is not particularly limited as long as it can maintain the shape of the culture solution holding chamber 10A. Examples of the material of the side wall 11 include polycarbonate (PC), polyester (PE), polystyrene (PS), glass, etc. The side wall 11 is preferably colorless and transparent, but may also be colored or not have light transmittance. When the side wall 11 is colored, it is preferably black to prevent autofluorescence and reflection.

[0060] The culture chip 20 is formed in a cylindrical shape with a top. The culture chip 20 has a frame 21 and a membrane member 22. The frame 21 abuts against the upper surface 12a of the bottom wall 12 of the container body 10. The lower surface 21b of the frame 21 abuts against the upper surface 12a of the bottom wall 12 by bonding. In addition, the frame 21 only needs to abut against the upper surface 12a of the bottom wall 12, and may be placed on the upper surface 12a of the bottom wall 12, or may be in a state of being closely attached to the upper surface 12a of the bottom wall 12 by van der Waals force, or may be bonded by an adhesive or the like.

[0061] Figure 2 It is a bottom view of the culture chip 20 according to the first embodiment.

[0062] Figure 2The culture chip 20 shown has a frame portion 21 that is rectangular when viewed from above. In addition, the shape of the frame portion 21 is not limited to a rectangular frame, as long as the culture chamber 4 can be formed inside it. In addition to the rectangular frame, for example, a polygonal frame, a circular frame, etc. can be cited. In addition, in the present embodiment, the frame portion 21 is formed into a square frame when viewed from above.

[0063] The size of the outer shape of the frame 21 can be any size as long as it can be arranged in the culture solution storage chamber 10A of the container body 10. For example, the width W1 of one side of the square outer edge of the frame 21 is in the range of 10 mm or more and 20 mm or less (for example, 15 mm). In addition, for example, the width W2 of one side of the square inner edge of the frame 21 is in the range of 2 mm or more and less than 10 mm (for example, 7 mm).

[0064] Back to Figure 1 The frame 21 has a height H1 for controlling the thickness of the culture tissue cultured in the culture chamber 4. The height H1 of the frame 21 is the dimension from its upper surface 21a to its lower surface 21b, and is preferably within a range of 50 μm to 200 μm when the culture object 3 is the testicular tissue of a mouse. In the embodiments described below, the testicular tissue of a mouse is used as the culture object 3, but the caliber of the seminiferous tubules of adult animals is about 350 μm in rats, about 200 μm in mice, about 300 μm in monkeys, and 220 μm to 250 μm in humans. Therefore, regardless of the animal, when the culture object 3 is the seminiferous tubules, it is preferred to set the height H1 of the frame 21 within a range of 50 μm to 350 μm to control the thickness of the seminiferous tubules. In addition, when the culture object 3 is a tissue other than mouse testis tissue, it is difficult to set the height below 20 μm, and when the culture object 3 is a large tissue, nutrients can be confirmed to enter the tissue by diffusion up to a height of 400 μm. Therefore, it is preferable to set the height H1 of the frame 21 within a range of 20 μm to 400 μm or less depending on the type of the culture object 3 to control the thickness of the culture object 3.

[0065] If the culture object 3 grows in size, oxygen ( Figure 1 Indicated by dashed arrows in the middle) and culture medium 2 ( Figure 1 On the other hand, by controlling the thickness of the culture object 3 using the height H1 of the frame 21, oxygen and the culture solution 2 can reach the center of the culture object 3, thereby preventing the center from being necrotized. In addition, the height H1 of the frame 21 can be set according to the type of the culture object 3.

[0066] The membrane member 22 has liquid permeability or liquid-containing substance permeability, and more preferably has light permeability. The material of the membrane member 22 is polycarbonate (PC) or polyethylene terephthalate (PET). In addition, as long as the membrane member 22 has liquid permeability or liquid-containing substance permeability, its material is not limited to polycarbonate or polyethylene terephthalate. For example, there are polytetrafluoroethylene (PTFE), nylon, nitrocellulose, polyvinylidene fluoride (PVDF), polyimide (PI), etc., as the form of the membrane member 22, it can also be a mesh membrane such as a porous membrane, a non-woven fabric, or a dialysis membrane.

[0067] The material of the membrane member 22 may be any material as long as it is non-toxic to the culture object 3 and can control the thickness of the culture object 3. As for the light transmittance of the membrane member 22, it is preferably colorless and transparent, and the membrane member 22 may have a liquid permeability that allows at least the culture solution 2 to pass through, or a liquid-containing substance permeability that allows the liquid substance in the culture solution 2, such as glucose, body fluid factors, and other liquid-containing substances to pass through. The lower surface 22b of the membrane member 22 is fixed to the upper surface 21a of the frame 21, and forms the culture chamber 4 together with the bottom wall 12 and the frame 21.

[0068] The material of the frame 21 is not particularly limited as long as it is non-toxic to the culture object 3 and can maintain the shape of the culture solution holding chamber 10A. The material of the frame 21 may be light-transmitting polydimethylsiloxane (PDMS) as in the bottom wall 12. In this case, the frame 21 and the membrane member 22 may be covalently bonded by silane coupling treatment.

[0069] In addition, the frame 21 may not have light transmittance, or may be a tape having adhesive surfaces on its upper surface 21a and lower surface 21b. That is, the frame 21 and the film member 22 may be fixed by an adhesive. As the tape forming the frame 21, a polyimide tape having a base material of polyimide (PI) and a silicone adhesive on its upper surface 21a and lower surface 21b is preferred.

[0070] According to the culture device 1 of the above structure, the culture object 3 can be cultured by preparing the container body 10, placing the culture object 3 and the culture chip 20 in order on the bottom wall portion 12 thereof, and introducing the culture solution 2 into the container body 10. The culture object 3 accommodated in the culture chamber 4 is supplied with oxygen ( Figure 1 ), the culture fluid 2 is supplied via the membrane member 22 ( Figure 1 Thus, the thickness of the culture object 3 can be controlled while observing the temporal and spatial morphological changes of the culture object 3.

[0071] Figure 3It is an explanatory diagram of the culture object observation method according to the first embodiment.

[0072] The method for observing a cultured object according to the present embodiment is, for example, Figure 3 As shown, the culture device 1 is placed on an inverted microscope 30 to observe the culture chamber 4 .

[0073] like Figure 1 As shown, the bottom wall 12 of the container body 10 has light permeability and air permeability in order to culture and observe the culture object 3. The culture chip 20 has a non-toxic sheet-shaped frame 21, and a film member 22 is bonded to the upper surface 21a of the frame 21. The film member 22 has light permeability like the bottom wall 12, so it does not hinder the observation of the inside of the culture chamber 4 through the bottom wall 12 by the inverted microscope 30.

[0074] The culture device 1 does not use agarose gel (agar) used in the conventional organ culture method, so the experimental preparation and operability are simpler than the conventional method. Furthermore, the culture chip 20 is easy to manufacture, and the range of options such as the culture range, the thickness of the sheet, and the type of the membrane member 22 is wide, so it is not limited to testis tissue, and can also be applied to the culture of other organ tissues, tissue slices (materials obtained by cutting tissues), cell blocks (organoids, spheroids, etc.), cells, bacteria, etc.

[0075] Thus, according to the culture device 1 of the present embodiment, high-precision optical observation can be performed using a high-power lens of an inverted microscope 30, and the culture device 1 can be directly set on the inverted microscope 30, so the operability is good compared with the previous method. Thus, for example, the temporal and spatial morphological changes of cells in sperm production can be captured in detail by real-time imaging, etc. Therefore, for example, machine learning (AI) can be used to analyze the nature of sperm production, which is helpful for drug development, medicine, life science research, etc. aimed at clarifying the treatment of sperm production disorders.

[0076] Thus, the culture device 1 of the present embodiment described above comprises a container body 10 for containing a culture solution 2, and a culture chip 20 forming a culture chamber 4 inside the container body 10, the container body 10 comprising a bottom wall portion 12 having light transmittance and air permeability, and the culture chip 20 comprising a frame portion 21 and a membrane member 22, the frame portion 21 being in contact with the upper surface 12a of the bottom wall portion 12, the membrane member 22 being liquid permeable or liquid-containing material permeable and fixed to the upper surface 21a of the frame portion 21, and forming the culture chamber 4 together with the bottom wall portion 12 and the frame portion 21. According to this configuration, the time resolution of observation can be improved without causing damage or contamination to the culture object 3.

[0077] In addition, in the present embodiment, the thickness of the culture object 3 accommodated in the culture chamber 4 is controlled by the height of the frame 21. According to this configuration, necrosis of the central portion of the culture object 3 can be suppressed.

[0078] In the present embodiment, the frame 21 is fixed to the upper surface 12a of the bottom wall 12. According to this configuration, positional deviation of the culture chip 20 in the culture solution storage chamber 10A and floating of the culture chip 20 can be suppressed.

[0079] In addition, in the present embodiment, the film member 22 has light transmissivity. According to this configuration, the culture object 3 can be easily observed in a bright field.

[0080] In addition, in this embodiment, the material of the bottom wall portion 12 is polydimethylsiloxane. According to this structure, it is possible to provide the bottom wall portion 12 with high light transmittance and air permeability.

[0081] In addition, in this embodiment, the material of the film member 22 is polycarbonate or polyethylene terephthalate. According to this structure, the film member 22 can be given high light permeability and liquid permeability.

[0082] In this embodiment, oxygen is supplied to the culture object 3 accommodated in the culture chamber 4 via the bottom wall 12, and the culture solution 2 is supplied via the membrane member 22. This configuration allows the temporal and spatial morphological changes of the culture object 3 to be observed while controlling the thickness of the culture object 3.

[0083] In this embodiment, the culture object 3 is a testis tissue. According to this configuration, it is possible to capture in detail the temporal and spatial morphological changes of cells during sperm production by real-time imaging or the like.

[0084] In addition, in the present embodiment, the frame portion 21 has a height H1 within a range of 50 μm to 200 μm. According to this configuration, necrosis of the central portion of the culture object 3 during culture can be suppressed.

[0085] In addition, the culture object observation method of this embodiment sets the culture device 1 on the inverted microscope 30, and observes the culture chamber 4 through the bottom wall portion 12. According to this structure, high-precision optical observation can be performed using the high-power lens of the inverted microscope 30, and the culture device 1 can be directly set on the inverted microscope 30, so the operability is improved compared with the conventional method.

[0086] In addition, the culture chip 20 of this embodiment includes: a membrane member 22 having liquid permeability or liquid-containing material permeability; and a frame 21 fixed to one surface (lower surface 22b) of the membrane member 22. According to this structure, it is easy to manufacture, and the range of options such as the culture range, the thickness of the sheet, and the type of the membrane member 22 is wide, so it is not limited to testis tissue, and can also be applied to the culture of other organ tissues, tissue slices (materials obtained by cutting tissues), cell masses (organoids, spheroids, etc.), cells, bacteria, etc.

[0087] [First embodiment]

[0088] The first embodiment of the present invention is described below. In addition, the present invention is not limited to the following first embodiment, and can be implemented by appropriately changing the scope of the present invention without changing its main purpose. In the following description, the same or equivalent configurations as those in the above-mentioned embodiment are marked with the same reference numerals, and the description thereof is simplified or omitted.

[0089] First, a comparative example to be compared with the first embodiment of the present invention will be described.

[0090] Figure 4 It is a diagram showing the structure of a culture apparatus 100 according to a comparative example.

[0091] like Figure 4 As shown, the culture device 100 of the comparative example includes a container body 110 , a culture chip 120 , and an agarose gel 130 .

[0092] The container body 110 has a culture solution storage chamber 110A formed inside thereof for storing the culture solution 2. The container body 110 is formed in a bottomed cylindrical shape, and has a side wall portion 111 and a bottom wall portion 112. An agarose gel 130 is placed on the bottom wall portion 112. The upper surface of the agarose gel 130 is located above the liquid surface of the culture solution 2, and supports the culture object 3. The reason why the culture object 3 is located above the liquid surface is that the culture object 3 will be necrotic if there is no supply of oxygen.

[0093] A culture chip 120 is placed on the upper surface of the agarose gel 130, and the culture chip 120 forms a culture chamber 4 for culturing the culture object 3. The culture chip 120 has light permeability and air permeability. Thus, oxygen can be supplied to the culture object 3 through the culture chip 120, and the culture solution 2 absorbed through the agarose gel 130 can be supplied to the culture object 3.

[0094] Figure 5 It is a diagram showing the results of a testis tissue culture experiment using the culture device 100 of the comparative example. Figure 6 It is a figure which shows the result of the testis tissue culture experiment using the culture apparatus 1 of the first embodiment.

[0095] In this culture experiment, testicular tissue removed from 7-day-old mice was used. Figure 5 and Figure 6 As shown, as the culture progressed, the thickness of the seminiferous tubules of the testicular tissue was observed to increase, confirming that good culture was possible.

[0096] Figure 7 This is a graph showing the postnatal age and seminiferous tubule growth rate of the testicular tissues of the first embodiment and the comparative example.

[0097] The seminiferous tubule growth rate is a value obtained by comparing the diameter of the seminiferous tubules in the culture using the first embodiment and the comparative example. Specifically, the diameter of the seminiferous tubules on the first day of culture is set as 100%, and the increase or decrease rate of the seminiferous tubule diameter is calculated every week. The result is as follows: Figure 7 As shown, it can be confirmed that the seminiferous tubules are thicker when using the culture device 1 of the first embodiment than when using the culture device 100 of the comparative example. This shows that the culture device 1 of the first embodiment can perform culture equal to or better than the conventional organ culture method using agarose gel 130.

[0098] Hereinafter, a preferred form of the film member 22 will be described.

[0099] Figure 8 This is a table summarizing the relationship between the pore diameter and the visibility of the culture object 3 when polycarbonate is used as the membrane member 22 . Fig. 9 This is a comparison photograph of the bright field and the fluorescent field (RFP) using an inverted microscope 30 when polycarbonate having a pore size of 0.4 μm is used as the membrane member 22 . Fig.10 This is a comparison photograph of the bright field and the fluorescence field (RFP) using an inverted microscope 30 when a polycarbonate having a pore size of 10 μm is used as the membrane member 22. Figure 8 and Fig.10 "S", "A", "B", and "C" shown in the figure represent the observability (observation ease) of the testicular tissue. Specifically, "S" represents the easiest observation, and the observation ease decreases in the order of "A", "B", and "C".

[0100] In addition, in the fluorescence field observation, the testis tissue taken out from the genetically modified mouse is used. The mouse is because the mitochondria in the sperm cell are marked by RFP (Red Fluorescent Protein, red fluorescent protein), so the evaluation of spermatogenesis can be carried out by fluorescence observation. In addition, the testis tissue of the genetically modified mouse that the acrosin in the sperm cell is marked by GFP (Green Fluorescent Protein, green fluorescent protein) can also be used.

[0101] like Fig. 9As shown in FIG. 1 , when polycarbonate having a pore size of 0.4 μm is used as the membrane member 22, the testicular tissue is hardly observed in the bright field, but the tissue can be observed in the fluorescence field based on RFP. Therefore, in this case, the observation ease of the testicular tissue is "C" in the bright field and "A" in the RFP (refer to FIG. Figure 8 ).

[0102] On the other hand, Fig.10 As shown in FIG. 1 , when polycarbonate with a pore size of 10 μm is used as the membrane member 22, the testicular tissue can be observed in both the bright field and the fluorescence field of RFP. Therefore, in this case, the ease of observing the testicular tissue is "A" in the bright field and "A" in the RFP (refer to FIG. Figure 8 ).

[0103] That is, when the material of the membrane member 22 is polycarbonate, the pore diameter is preferably 10 μm or more from the viewpoint of easy observation.

[0104] Fig.11 This is a table summarizing the relationship between the pore diameter and the visibility of the culture object 3 when polyethylene terephthalate is used as the membrane member 22 . Fig.12 This is a comparison photograph of a bright field and a fluorescent field (RFP) using an inverted microscope 30 when polyethylene terephthalate having a pore size of 0.45 μm is used as the film member 22 . Fig.13 This is a comparison photograph of the bright field and the fluorescent field (RFP) using an inverted microscope 30 when polyethylene terephthalate having a pore size of 3 μm is used as the film member 22 .

[0105] like Fig.12 As shown in FIG. 1 , when polyethylene terephthalate having a pore size of 0.45 μm is used as the membrane member 22, the testicular tissue can be clearly observed in both the bright field and the fluorescence field of RFP. Therefore, in this case, the ease of observing the testicular tissue is "S" in the bright field and "A" in the RFP (refer to FIG. Fig.11 ).

[0106] On the other hand, Fig.13 As shown in FIG. 1 , when polyethylene terephthalate with a pore size of 3 μm is used as the membrane member 22, although the testicular tissue can be observed in the bright field and the fluorescence field of RFP, many pores are reflected. Therefore, in this case, the observation ease of the testicular tissue is "B" in the bright field and "A" in the RFP (refer to FIG. Fig.11 ).

[0107] As described above, when the material of the film member 22 is polyethylene terephthalate, the pore diameter is preferably 0.45 μm from the viewpoint of easy observation.

[0108] Fig.14 This is a table summarizing the materials, pore sizes, porosity, and light transmittance of the film member 22 used in the first embodiment.

[0109] When bright field observation based on transmitted light is performed using an inverted microscope 30, the light transmittance of the film member 22 varies depending on the pore size and porosity of the film member 22 used. Fig.14 As shown, the polycarbonate film member 22 with a pore size of 0.4 μm is turbid and cannot provide sufficient light transmittance required for optical observation. The polyethylene terephthalate film member 22 with a pore size of 3 μm is slightly whitish and is not unobservable for optical observation, but is not optimal.

[0110] On the other hand, the other film members 22 (polycarbonate with a thickness of 10 μm, polyethylene terephthalate with a pore size of 0.45 μm) are colorless and transparent and have sufficient light transmittance.

[0111] When observing using an inverted microscope 30 using fluorescence observation based on reflected light, i.e. RFP, the excitation light for observation is irradiated from the bottom wall 12 side, so the ease of observation is always "A" regardless of the light transmittance of the membrane member 22 and the pore size of 0.4μm to 10μm.

[0112] When observing in bright field, the light for observation is irradiated from the side of the film component 22, so the light transmittance of the film component 22 will be greatly affected. Therefore, in the film component 22 of polycarbonate with a pore size of 10μm and polyethylene terephthalate with a pore size of 0.45μm that can fully obtain light transmittance, the ease of observation becomes "A" and "S".

[0113] Furthermore, since the culture object 3 can be cultured sufficiently regardless of the pore size, porosity, light transmittance, etc. of the membrane member 22, when bright field observation is performed, a membrane member 22 having excellent light transmittance may be selected.

[0114] (Second Embodiment)

[0115] Next, a second embodiment of the present invention will be described. In the following description, the same reference numerals are attached to the same or equivalent components as those in the above-described embodiment, and the description thereof will be simplified or omitted.

[0116] In the culture device 1 of the second embodiment, Figure 1 The bottom wall portion 12 shown in the figure is made of a 4-methyl-1-pentene polymer, which is different from the above-mentioned embodiment.

[0117] 4-methyl-1-pentene polymer has light transmittance and air permeability like the above-mentioned polydimethylsiloxane (PDMS), but is superior to polydimethylsiloxane in the following aspects. First, 4-methyl-1-pentene polymer is harder than polydimethylsiloxane and is easy to maintain the shape of the bottom wall 12. In addition, compared with polydimethylsiloxane, 4-methyl-1-pentene polymer has low adsorption of drugs and culture fluid components and moderately permeates oxygen. Therefore, 4-methyl-1-pentene polymer is suitable as the material of the bottom wall 12 of the culture device 1. In addition, if the oxygen permeability of the bottom wall 12 is too low, the cell tissue of the culture object 3 cannot grow fully. On the contrary, if the oxygen permeability is too high, the cell tissue of the culture object 3 is hypertrophied and the cells are crushed to death in the culture chamber 4. Therefore, it is better that the bottom wall 12 is moderately permeated with oxygen.

[0118] Specifically, the oxygen permeability of the bottom wall portion 12 at a temperature of 23°C and a humidity of 0% is 4500 to 90000 cm 3 / (m 2 ×24h×atm), preferably 4500~67500cm 3 / (m 2 ×24h×atm), more preferably 4500~47000cm 3 / (m 2 ×24h×atm), more preferably 4500 to 45000cm 3 / (m 2 ×24h×atm).

[0119] Moreover, although the detailed reasons are not yet clear, by using 4-methyl-1-pentene polymer as the material of the bottom wall portion 12, the culture function of the culture device 1 is improved compared to the case where other breathable materials such as polydimethylsiloxane, tetrafluoroethylene-hexafluoropropylene copolymer (FEP) are used, and the culture object 3 (especially testicular tissue) can be well cultured.

[0120] In the present embodiment, a 4-methyl-1-pentene homopolymer and a copolymer of 4-methyl-1-pentene and other monomers are collectively referred to as a "4-methyl-1-pentene-based polymer".

[0121] As an example of a 4-methyl-1-pentene polymer, a copolymer of 4-methyl-1-pentene and other monomers may be any of a random copolymer, an alternating copolymer, a block copolymer, and a graft copolymer. As a copolymer of 4-methyl-1-pentene and other monomers, a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and an α-olefin having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) is preferred because it has high strength and is not easily torn or broken even when used as a material for the bottom wall portion 12, and has little deflection.

[0122] The 4-methyl-1-pentene polymer is preferably at least one polymer selected from a 4-methyl-1-pentene homopolymer and a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), and more preferably a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

[0123] As the aforementioned olefin, ethylene, propylene, 1-butene, 1-hexene, 1-heptene, 1-octene, 1-decene, 1-tetradecene, 1-hexadecene, 1-heptadecene, 1-octadecene and 1-eicosene can be mentioned. The aforementioned olefin can be appropriately selected according to the physical properties required for the culture member. For example, as the aforementioned olefin, from the viewpoint of appropriate oxygen permeability and excellent rigidity, α-olefins with 8 to 18 carbon atoms are preferred, and at least one selected from 1-octene, 1-decene, 1-dodecene, 1-tetradecene, 1-hexadecene, 1-heptadecene and 1-octadecene is more preferred. When the carbon number of the olefin is within the above range, the processability of the polymer becomes better, and there is a tendency that the appearance of the bottom wall portion 12 caused by cracks and ruptures of the end is not easy to occur. In addition, the defective product generation rate of the bottom wall portion 12 becomes low.

[0124] The aforementioned olefins may be used in one or more kinds. From the viewpoint of material strength, the number of carbon atoms is preferably 2 or more, and more preferably 10 or more. In the case of combining two or more different α-olefins, it is particularly preferred to combine at least one selected from 1-tetradecene and 1-hexadecene with at least one selected from 1-heptadecene and 1-octadecene.

[0125] The content of the structural unit derived from 4-methyl-1-pentene in the 4-methyl-1-pentene polymer is preferably 60 to 100 mol%, more preferably 80 to 98 mol%.

[0126] In addition, when the 4-methyl-1-pentene polymer is a copolymer of 4-methyl-1-pentene and at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene), the content of the structural unit derived from at least one olefin selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene) in the copolymer is preferably 0 to 40 mol%, more preferably 2 to 20 mol%. In addition, regarding the content of these structural units, the amount of all repeating structural units in the 4-methyl-1-pentene polymer is set to 100 mol%. If the content of the structural unit is within the above range, a homogeneous bottom wall portion 12 with excellent processability can be obtained, and the balance between the toughness and strength of the film is good, so the deflection is also reduced.

[0127] The aforementioned 4-methyl-1-pentene polymer may have structural units other than structural units derived from 4-methyl-1-pentene and structural units derived from the aforementioned ethylene and α-olefins having 3 to 20 carbon atoms (hereinafter also referred to as "other structural units") within a range that does not impair the effects of the present invention. The content of the other structural units is, for example, 0 to 10.0 mol%. When the aforementioned 4-methyl-1-pentene polymer has other structural units, the other structural units may be one type or two or more types.

[0128] As monomers for introducing other structural units, for example, cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins can be cited. As cyclic olefins, aromatic vinyl compounds, conjugated dienes, non-conjugated polyenes, functional vinyl compounds, hydroxyl-containing olefins, and halogenated olefins, for example, compounds described in paragraphs

[0035] to

[0041] of Japanese Patent Application Laid-Open No. 2013-169685 can be used.

[0129] The 4-methyl-1-pentene polymer may be used alone or in combination of two or more.

[0130] As 4-methyl-1-pentene polymer, commercially available products can also be used. Specifically, TPX MX001, MX002, MX004, MX0020, MX021, MX321, RT18, RT31 or DX845 (all trademarks) made by Mitsui Chemicals (Strain) can be cited. In addition, even if it is manufactured by other manufacturers, as long as it is a 4-methyl-1-pentene polymer that meets the above requirements, it can also be preferably used. These commercial products can be used alone or in combination of two or more.

[0131] 4-methyl-1-pentene polymers generally have a melting point of 200°C to 240°C and are highly heat resistant. In addition, since hydrolysis does not occur, water resistance, boiling water resistance, and steam resistance are excellent, so a culture device having a bottom wall portion containing 4-methyl-1-pentene polymers can be sterilized by high-pressure steam. 4-methyl-1-pentene polymers also have the characteristics of high visible light transmittance (usually 90% or more) and no autofluorescence, so a culture device 1 having a bottom wall portion 12 containing 4-methyl-1-pentene polymers is easy to observe the culture object 3. Furthermore, since it shows excellent chemical resistance to most chemicals and is difficult to adsorb drugs, it is also suitable for drug development screening purposes and diagnostic purposes. 4-methyl-1-pentene polymers can be heat-sealed, and not only the heat fusion of the materials themselves is easy, but also the heat bonding with other materials is easy. In addition, since it can be thermoformed, it is easy to form into any shape, such as using an embossing method or an insert method.

[0132] The weight average molecular weight (Mw) of the 4-methyl-1-pentene polymer measured by gel permeation chromatography (GPC) using standard polystyrene as a reference substance is preferably 10,000 to 2,000,000, more preferably 20,000 to 1,000,000, and further preferably 30,000 to 500,000. Here, the sample concentration during GPC measurement can be set to, for example, 1.0 to 5.0 mg / ml. In addition, the molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer is preferably 1.0 to 30, more preferably 1.1 to 25, and further preferably 1.1 to 20. The solvent used in GPC is preferably o-dichlorobenzene. In addition, as an example of the measurement conditions, the conditions shown below can be cited, but are not limited to the measurement conditions.

[0133] <Measurement Conditions of Weight Average Molecular Weight (Mw) and Molecular Weight Distribution (Mw / Mn)>

[0134] The weight average molecular weight (Mw) and number average molecular weight (Mn) of the polymer (4-methyl-1-pentene polymer) dissolved in o-dichlorobenzene were measured under the following conditions by calibrating the molecular weight using standard polystyrene.

[0135] · Apparatus: Gel Permeation Chromatograph HLC-8321GPC / HT (manufactured by Tosoh Corporation)

[0136] Data analysis software: Empower3 (manufactured by Waters)

[0137] Detector: Differential refractometer

[0138] Columns connected in series: TSKgel GMH6-HT (2 columns) and TSKgel GMH6-HTL (2 columns)

[0139] Column temperature: 140℃

[0140] Flow rate: 1.0ml / min

[0141] · Sample concentration: 1.5mg / ml

[0142] By setting the weight average molecular weight (Mw) to below the above upper limit, when the 4-methyl-1-pentene polymer is formed, the film produced by melt forming is easy to suppress the occurrence of adverse conditions such as gel, and is easy to make a film with uniform surface. In addition, when produced by a solution casting method, the solubility in a solvent is made better, and adverse conditions such as gel of the film are easily suppressed, and is easy to make a film with uniform surface.

[0143] In addition, by setting the weight average molecular weight (Mw) to be above the above lower limit, the bottom wall portion 12 tends to have sufficient strength. In addition, by setting the molecular weight distribution within the above range, it is easy to suppress the surface stickiness of the bottom wall portion 12, and the toughness tends to be sufficient, and it is easy to suppress the occurrence of bending and cracks.

[0144] The weight average molecular weight (Mw) and molecular weight distribution (Mw / Mn) of the 4-methyl-1-pentene polymer may be such that, when two or more 4-methyl-1-pentene polymers are used, their respective Mw and Mw / Mn are within the above ranges.

[0145] Since 4-methyl-1-pentene polymers have the excellent properties described above, the culture device 1 in which at least the bottom wall portion 12 is formed of the 4-methyl-1-pentene polymer will not have an adverse effect on the culture, and has good stability and light transmittance and can be sterilized, and is therefore very excellent.

[0146] The method for producing the above-mentioned 4-methyl-1-pentene polymer can be any method as long as 4-methyl-1-pentene, olefins, and other monomers can be polymerized. In addition, in order to control the molecular weight and molecular weight distribution, a chain transfer agent such as hydrogen can be coexisted. There is no limitation on the equipment used in the production. The polymerization method can be a known method, or it can be a gas phase method, a slurry method, a solution method, or a bulk method. Preferred are a slurry method and a solution method. In addition, the polymerization method can be a method in which a plurality of polymers with different molecular weights are blended into a polymerization system by a single-stage polymerization method or a multi-stage polymerization method such as a two-stage polymerization method. Regardless of whether it is a single-stage or multi-stage polymerization method, when hydrogen is used as a chain transfer agent, it can be added all at once or in batches, for example, it can be added at the initial, middle, or final stage of the polymerization. The polymerization can be carried out at room temperature or heated as needed. From the viewpoint of polymerization efficiency, it is preferably carried out at 20°C to 80°C, and particularly preferably at 40°C to 60°C. The catalyst used in the production is also not limited. From the viewpoint of polymerization efficiency, it is preferred to use, for example, the solid titanium catalyst component (I) described in International Publication No. 2006 / 054613 or the olefin polymerization catalyst (metallocene catalyst) containing a transition metal compound (A) described in International Publication No. 2014 / 050817.

[0147] In addition, when the bottom wall portion 12 is formed of a composition containing a 4-methyl-1-pentene polymer, the 4-methyl-1-pentene polymer preferably accounts for 90% by mass or more and less than 100% by mass, more preferably 95% by mass or more and less than 100% by mass, and particularly preferably 99% by mass or more and less than 100% by mass, in 100% by mass of the composition forming the bottom wall portion 12. If a large amount of components other than the 4-methyl-1-pentene polymer is contained, not only the oxygen permeability is reduced, but also the transparency and strength are reduced.

[0148] The material forming the bottom wall portion 12 may also contain components other than the 4-methyl-1-pentene polymer. As components other than the 4-methyl-1-pentene polymer, additives such as heat stabilizers, light stabilizers, processing aids, plasticizers, antioxidants, lubricants, defoamers, anti-blocking agents, colorants, modifiers, antibacterial agents, antifungal agents, and antifogging agents can be cited.

[0149] [Second embodiment]

[0150] Hereinafter, a second embodiment of the present invention will be described. Note that, similarly to the first embodiment, the present invention is not limited to the second embodiment described below, and can be implemented with appropriate changes within the scope of the gist thereof.

[0151] Fig.15 It is a graph showing the postnatal age and Acr-GFP expression rate of the testicular tissues of the second example and the comparative example.

[0152] The Acr-GFP expression rate is obtained by the following formula (1), where the fluorescent field area of ​​the testis tissue of a genetically modified mouse in which acrosin in sperm cells is labeled with GFP (Green Fluorescent Protein) is defined as A1 and the bright field area of ​​the testis tissue is defined as A2.

[0153] Acr-GFP expression rate (%) = A1 / A2…(1)

[0154] In the second embodiment, the height of the testis tissue is controlled to 85 μm using the culture chip 20. In addition, in the second embodiment, polydimethylsiloxane (PDMS), TPX (registered trademark) as a 4-methyl-1-pentene polymer (manufactured by Mitsui Chemicals, Inc.: molecular weight (Mw) = 428,000, molecular weight distribution (Mw / Mn) = 4.1), and Lumox (registered trademark) as a tetrafluoroethylene-hexafluoropropylene copolymer (FEP) (manufactured by SARSTEDT Co., Ltd.) are used as the material of the bottom wall portion 12. In addition, as a comparative example, Figure 4 The culture device 100 is shown.

[0155] Fig.15 The graph shown shows the experimental results when the thickness of the bottom wall 12 is set to 500 μm and the number of samples is set to 6 in the case of polydimethylsiloxane (PDMS). In addition, in the case of 4-methyl-1-pentene polymer (TPX), the graph shows the experimental results when the thickness of the bottom wall 12 is set to 50 μm and the oxygen permeability is set to 38240 cm at a temperature of 23° C. and a humidity of 0%. 3 / (m 2 ×24h×atm), and the number of samples was set to 12. In the case of TPX, the thickness of the bottom wall 12 is 1 / 10 of that of polydimethylsiloxane, but the oxygen permeability of TPX is approximately 1 / 10 of that of polydimethylsiloxane, so the conditions are approximately the same. In the case of Lumox, the experimental results are shown when the thickness of the bottom wall 12 is 25 μm and the number of samples is 2. In the case of Lumox (registered trademark), the conditions are also set to be approximately the same in consideration of the relationship between the bottom wall 12 and the oxygen permeability.

[0156] like Fig.15 As shown, when the material of the bottom wall 12 is 4-methyl-1-pentene polymer (TPX), it is found that the Acr-GFP expression rate is superior to that of the bottom wall 12 made of polydimethylsiloxane (PDMS), Lumox, and the comparative example.

[0157] Fig.16This is a diagram comparing fluorescent images of testicular tissue at postnatal age (35th day) in a comparative example and when polydimethylsiloxane (PDMS) is used as the bottom wall portion 12 . Fig.17 The figure compares the fluorescent images of the testicular tissue at postnatal age (35th day) when 4-methyl-1-pentene polymer (TPX) and Lumox (registered trademark) are used as the bottom wall portion 12 .

[0158] like Fig.16 and Fig.17 As shown, at the postnatal age (35th day), when 4-methyl-1-pentene polymer (TPX) was used as the bottom wall 12, Acr-GFP was clearly expressed in the testis tissue compared with other materials or comparative examples. This shows that according to the culture device 1 of the second embodiment, when 4-methyl-1-pentene polymer (TPX) was used as the bottom wall 12, Acr-GFP expression was excellent and fluorescence observation was easy compared with the case of using 4-methyl-1-pentene polymer (TPX), Lumox, and the comparative example.

[0159] (Third Embodiment)

[0160] Next, a third embodiment of the present invention will be described. In the following description, the same reference numerals are attached to the same or equivalent components as those in the above-described embodiment, and the description thereof will be simplified or omitted.

[0161] Fig.18 This is a diagram showing a state where a culture solution 2 and a culture object 3 are placed in a culture device 1 according to a third embodiment.

[0162] The upper surface 12a of the bottom wall 12 of the culture device 1 of the third embodiment is hydrophobic. For example, when the material of the bottom wall 12 is a 4-methyl-1-pentene polymer, the bottom wall 12 itself is hydrophobic. That is, the entire upper surface 12a of the bottom wall 12 becomes a hydrophobic region 40. In addition, the "hydrophobicity" mentioned here means that the water contact angle of the upper surface 12a of the bottom wall 12 is 80° or more and 160° or less, preferably 90° or more and 160° or less, more preferably more than 100° and 150° or less, and further preferably has a parameter of 105° or more and 130° or less.

[0163] In addition, the method for measuring the water contact angle is not particularly limited, and a known method can be used, preferably a sessile drop method. The water contact angle can be measured, for example, by the following method: according to Japanese Industrial Standard JIS-R3257 (Wettability Test Method for Substrate Glass Surface), under constant temperature and humidity conditions of 25±5°C and 50±10%, a water drop of a volume of 4 μL or less, in which the shape of the water drop is regarded as a sphere, is added to the surface of a culture member or a measurement sample made of the same material as the culture member, and the angle of the contact interface between the measurement sample and the water drop within 1 minute after the water drop just contacts the measurement sample surface is measured by the sessile drop method.

[0164] Fig.18 (a) shows a first step of dripping a culture solution 2 onto an upper surface 12a of a bottom wall portion 12 of a container body 10 having an air-permeable bottom wall portion 12 to form droplets. Fig.18 (a) shows the second step of sealing the culture object 3 in the droplet. Fig.18 As shown in (a), by dripping the culture solution 2 onto the upper surface 12a of the bottom wall 12, liquid droplets (micron-order tiny water droplets) can be easily prepared. Thus, the culture object 3 can be easily enclosed in the culture solution 2.

[0165] Fig.18 (b) shows a third step of providing a frame 21 abutting against the upper surface 12a of the bottom wall 12 and a membrane member 22 fixed to the upper surface 21a of the frame 21, and forming a culture chamber 4 for accommodating the culture object 3 by the bottom wall 12, the frame 21 and the membrane member 22, wherein the frame 21 surrounds the droplets enclosing the culture object 3, and the membrane member 22 has liquid permeability or liquid-containing material permeability. By forming the droplets of the culture solution 2 as described above, when the culture chip 20 is set on the bottom wall 12, it is easy to fill the culture chamber 4 with the culture solution 2.

[0166] Fig.19 This is a diagram showing a state where a culture solution 2 and a culture object 3 are placed in a culture device 1 in which an upper surface 12a of a bottom wall portion 12 is made hydrophilic.

[0167] Fig.19 The upper surface 12a of the bottom wall portion 12 shown in (a) is covered with a hydrophilic coating layer 13. That is, the entire upper surface 12a of the bottom wall portion 12 becomes a hydrophilic region 50. In addition, "hydrophilicity" refers to a parameter other than the above-mentioned "hydrophobicity".

[0168] If this is the composition, then Fig.19 As shown in (a), even if the culture solution 2 is dripped onto the upper surface 12a of the bottom wall portion 12, the culture solution 2 will wet and spread, and no droplets can be formed. Fig.19As shown in (b), when the culture chip 20 is set on the bottom wall 12, air is likely to remain in the culture chamber 4. In addition, the culture solution 2 will penetrate into the lower surface 21b of the frame 21, making it difficult to set (bond) the culture chip 20 on the bottom wall 12.

[0169] On the other hand, when the culture solution 2 is supplied to the culture solution storage chamber 10A, when the upper surface 12a of the bottom wall 12 is made hydrophilic, the amount of the culture solution 2 supplied can be reduced compared to when the upper surface 12a of the bottom wall 12 is made hydrophobic. Specifically, when the upper surface 12a of the bottom wall 12 is made hydrophobic, Fig.18 As shown in (c), in order to immerse the culture chip 20 in the culture solution 2, the liquid level L1 is required. On the other hand, when the upper surface 12a of the bottom wall portion 12 is made hydrophilic, as shown in FIG. Fig.19 As shown in (c) , in order to immerse the culture chip 20 in the culture solution 2, a liquid level L2 lower than the liquid level L1 is sufficient.

[0170] Therefore, the following Fig. 20 and Fig.21 The configuration shown is preferred.

[0171] Fig. 20 This is a diagram showing a state where a culture solution 2 and a culture object 3 are placed in a culture device 1 according to a modification of the third embodiment. Fig.21 yes Fig. 20 A top view of the container body 10 is shown.

[0172] As shown in these figures, in the bottom wall 12 , the portion forming the culture chamber 4 on the upper surface 12 a of the bottom wall 12 is hydrophobic (hydrophobic region 40 ), and the portion other than the portion forming the culture chamber 4 is hydrophilic (hydrophilic region 50 ).

[0173] According to the above composition, if Fig. 20 As shown in (a), by dripping the culture solution 2 on the hydrophobic area 40 of the bottom wall 12, it is easy to make droplets and to seal the culture object 3 in the droplets. Fig. 20 As shown in (b), when the culture chip 20 is set on the bottom wall 12, it is easy to fill the culture chamber 4 with the culture solution 2. In addition, the culture solution 2 does not wet and spread, so it is easy to set the culture chip 20 on the bottom wall 12. Fig. 20 As shown in (c), only the liquid level L2 is sufficient to immerse the culture chip 20 in the culture solution 2. Thus, sufficient cell culture can be performed with a small amount of culture solution 2, so the running cost is reduced.

[0174] In addition, if Fig.21As shown, the outer shape of the culture chip 20 (frame 21, membrane member 22) is rectangular, but the inner shape of the frame 21 forming the culture chamber 4 is preferably circular. If the inner shape of the frame 21 is circular, air is less likely to remain in the corners compared to the case where the inner shape of the frame 21 is a quadrilateral, and when the culture chip 20 is set, it is easy to fill the culture chamber 4 with the culture solution 2. In addition, if the outer shape of the culture chip 20 is rectangular, it can be mass-produced by cutting sheets or the like, and the culture chip 20 can be manufactured at a low cost.

[0175] In another variation of the third embodiment, a part or all of the upper surface 12a of the bottom wall portion 12 may be surface treated or covered with a coating layer different from the above-mentioned hydrophilic coating layer 13, thereby forming a specific area. The water contact angle of the above-mentioned specific area formed on the upper surface 12a of the bottom wall portion 12 can be set to, for example, 30° or more and 160° or less.

[0176] The preferred embodiments of the present invention are described and explained above, but it should be understood that these are illustrative embodiments of the present invention and should not be considered as limiting the embodiments of the present invention. Additions, omissions, substitutions and other changes can be made without departing from the scope of the present invention. Therefore, the present invention should not be considered as being limited by the above description, but by the claims.

[0177] [Note]

[0178] Furthermore, the above-mentioned culture device 1 is excellent not only as a method for observing a culture object, but also as a method for culturing a culture object 3 (for example, see Fig.15 When the purpose is to culture the culture object 3, the bottom wall portion 12 does not necessarily need to be light-transmissive.

[0179] Therefore, part or all of the above-mentioned embodiments may be described as follows with respect to the cultivation of the culture object 3, particularly the cultivation of testicular tissue, as the subject and purpose, but are not limited to the following.

[0180] (Note 1)

[0181] A culture device comprises a container body for accommodating a culture solution, and a culture chip forming a culture chamber inside the container body.

[0182] The container body has a bottom wall portion having air permeability.

[0183] The aforementioned culture chip has:

[0184] a frame portion abutting against the upper surface of the bottom wall portion, and

[0185] The membrane member is liquid-permeable or liquid-containing material-permeable and is fixed to the upper surface of the frame portion, and forms the culture chamber together with the bottom wall portion and the frame portion.

[0186] (Note 2)

[0187] The culture device according to (Supplementary Note 1), wherein the material of the bottom wall portion is a 4-methyl-1-pentene polymer.

[0188] (Note 3)

[0189] The culture device according to (Supplementary Note 2), wherein the 4-methyl-1-pentene-based polymer is a copolymer of 4-methyl-1-pentene and at least one selected from ethylene and α-olefins having 3 to 20 carbon atoms (excluding 4-methyl-1-pentene).

[0190] (Note 4)

[0191] The culture device according to any one of (Supplementary Note 1) to (Supplementary Note 3), wherein the oxygen permeability of the bottom wall portion at a temperature of 23° C. and a humidity of 0% is 4500 to 90000 cm 3 / (m 2 ×24h×atm).

[0192] (Note 5)

[0193] The culture device according to any one of (Supplementary Note 1) to (Supplementary Note 4), wherein the bottom wall portion has light translucency.

[0194] (Note 6)

[0195] A cell culture method using the culture device described in any one of (Supplement 1) to (Supplement 5), wherein oxygen in the gas phase is supplied to a culture object contained in the culture chamber via the bottom wall portion, and the culture solution is supplied via the membrane member.

[0196] (Note 7)

[0197] A cell culture method, comprising at least:

[0198] In the first step, a culture solution is dripped onto an upper surface of a bottom wall of a container body having a bottom wall to form droplets, wherein the bottom wall is air-permeable;

[0199] The second step is to seal the culture object in the aforementioned droplet; and

[0200] In the third step, a frame portion abutting against the upper surface of the aforementioned bottom wall portion and a membrane component fixed to the upper surface of the aforementioned frame portion are provided, and a culture chamber for accommodating the aforementioned culture object is formed by the aforementioned bottom wall portion, the aforementioned frame portion and the aforementioned membrane component, wherein the aforementioned frame portion surrounds the aforementioned liquid droplets enclosing the aforementioned culture object, and the aforementioned membrane component is liquid-permeable or liquid-containing substance-permeable.

[0201] Furthermore, the components in the above-described embodiments may be appropriately replaced with known components without departing from the spirit of the present invention, and the above-described modifications may be appropriately combined.

[0202] Description of Reference Numerals

[0203] 1···culture device, 2···culture solution, 3···culture object, 4···culture chamber, 10···container body, 10A···culture solution storage chamber, 11···side wall portion, 11a···upper surface, 11b···lower surface, 12···bottom wall portion, 12a···upper surface, 20···culture chip, 21···frame portion, 21a···upper surface, 21b···lower surface, 22···membrane member, 22b ···lower surface, 30···inverted microscope, 40···hydrophobic area, 50···hydrophilic area, 100···culture device, 110···container body, 110A···culture fluid holding chamber, 111···side wall, 112···bottom wall, 120···culture chip, 130···agarose gel, H1···height, L1···liquid level, L2···liquid level, W1···width, W2···width.

Claims

1. A culture device comprising a container body for containing a culture solution, and a culture chip forming a culture chamber inside the container body, The container body includes a bottom wall portion having light-transmitting and air-permeable properties. The culture chip has: a frame portion abutting against the upper surface of the bottom wall portion, and The membrane member is liquid-permeable or liquid-containing material-permeable and is fixed to the upper surface of the frame portion, and forms the culture chamber together with the bottom wall portion and the frame portion.

2. The culture device according to claim 1, wherein: To the culture object accommodated in the culture chamber, oxygen in the gas phase is supplied via the bottom wall portion, and the culture solution is supplied via the membrane member.

3. The culture device according to claim 1, wherein: The thickness of the culture object accommodated in the culture chamber is controlled by the height of the frame.

4. The culture device according to claim 1, wherein: The film member has light translucency.

5. The culture device according to claim 1, wherein: The frame portion is fixed to the upper surface of the bottom wall portion.

6. The culture device according to claim 1, wherein: The frame portion has a height within a range of 20 μm or more and 400 μm or less.

7. The culture device according to claim 2, wherein: The culture object is a seminiferous tubule, and the frame has a height within a range of 50 μm to 350 μm.

8. The culture device according to claim 2, wherein: The culture object is a mouse testis tissue, and the frame has a height within a range of 50 μm to 200 μm.

9. The culture device according to claim 1, wherein: The upper surface of the bottom wall portion is hydrophobic.

10. The culture device according to claim 1, wherein: A portion of the upper surface of the bottom wall portion that forms the culture chamber is hydrophobic, and a portion other than the portion that forms the culture chamber is hydrophilic. 11 . A method for observing a culture object, comprising installing the culture device according to claim 1 on an inverted microscope and observing the culture chamber through the bottom wall portion.

12. A culture chip, comprising: a membrane member having liquid permeability or liquid-containing material permeability, and A frame portion is fixed to one surface of the membrane member.

13. The culture chip according to claim 12, wherein: The film member has light translucency.

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