TWI713598B - Seal for display of culture vessel - Google Patents

Abstract

The subject of the present invention is to provide a fertilized egg or cell culture method or culture kit, even if attached to the surface of a culture container for mammalian fertilized egg or cell culture, it will not affect the embryogenesis of the fertilized egg or the proliferation of mammalian cells/ Seals for displaying bad effects on survival may not have bad effects on the embryogenesis or cell proliferation/survival of the fertilized egg. The embryos of the mammalian blastoderm sac can be made to culture mammalian cells. A water-soluble adhesive layer is formed on one side of the substrate layer to reduce or inhibit the generation of outgas, because it will not adversely affect the embryogenesis of fertilized eggs or the proliferation/survival of mammalian cells. Therefore, in vitro culture of fertilized eggs in a culture container attached with such a water-soluble adhesive seal can efficiently produce good embryos of the blastocyst.

Description

Seal for display of culture vessel

The present invention relates to a display seal attached to the surface of a culture container used for mammalian fertilized eggs or mammalian cell culture, and a method for culturing fertilized eggs or cells cultured in a culture container attached with such a display seal, Or a kit for culturing mammalian fertilized eggs or mammalian cells equipped with the above-mentioned display seal.

In Japan, due to late marriage or late childbirth, one group of couples in the four groups has infertility problems. Therefore, in addition to general infertility treatment by artificial insemination (AIH), the implementation of advanced infertility treatment (ART) such as in vitro fertilization (IVF) and micro insemination (ICSI) tends to increase.

Now, in vitro fertilization of sperm and eggs cultured under the condition of the oviduct close to the original fertilization site. After making a fertilized egg, it can pass through the stages of cleavage, morula, and germinal sac until it hatches from the yolk membrane. The blastocyst stage is cultured. Although embryos are transferred to the uterus at the stage of the blastocyst, a high pregnancy rate is obtained, but the rate of embryos that develop until the stage of the blastocyst is 40-50%. In particular, the rate of a good blastocyst is as low as 20-30% becomes a problem.

The in vitro culture of fertilized eggs is usually carried out by a method (microdroplet method) in which a droplet of 20-30 μL is formed on a dish, and a single or multiple fertilized eggs are placed in the droplet for culture. In order to improve such a micro-droplet method, a culture container is proposed in which the operability of the fertilized egg in the droplet with a dropper is improved, and stable culture liquid droplets can be formed (Patent Document 1). In addition, a method has also been proposed in which fertilized eggs and endometrial cells are co-cultured in an environment closer to the living body to improve the generation efficiency (Patent Document 2).

On the other hand, since the trays for culturing the patient’s embryos are managed individually, although the markers are recorded or the seal samples are identified, it does not take into account the impact of the seals attached to the culture trays on the fertilized eggs. Possibility of bad effects on embryogenesis. Prior art documents Patent documents

Patent Document 1 JP 2015-29431 Bulletin Patent Document 2 JP 2012-75391 Bulletin

The problem to be solved by the invention

The subject of the present invention is to provide a fertilized egg or cell culture method or culture kit, even if attached to the surface of a culture container for mammalian fertilized egg or cell culture, it will not affect the embryogenesis of the fertilized egg or the proliferation of mammalian cells/ Seals for displaying bad effects on survival may not have bad effects on the embryogenesis or cell proliferation/survival of the fertilized egg. The embryos of the mammalian blastoderm sac can be made to culture mammalian cells. Means to solve the problem

As mentioned above, in the in vitro culture of fertilized eggs, the display seal attached to the surface of the culture container has a bad effect on the embryogenesis of the fertilized eggs, which is not known to those in the art. The inventors of this case, as a result of research using a vented seal and a less vented seal (water-soluble adhesive seal), they found that venting (at least 2-propanol) has a bad effect on the embryogenesis of fertilized eggs. And the water-soluble adhesive seal that inhibits or reduces the exhaust gas will not have a bad influence on the embryogenesis of the fertilized egg. Therefore, the fertilized egg can be cultured in vitro in a culture container attached with such a water-soluble adhesive seal, which can be efficiently produced. Embryos of human and other mammalian blastocysts have completed the present invention.

That is, the present invention is as described below.

[1] A display seal for attaching to the surface of a culture container used for mammalian fertilized eggs or cell culture, characterized in that a water-soluble adhesive layer is formed on one side of the substrate layer to reduce or inhibit gas out (Outgas) produced.

[2] The display seal as described in [1] above, wherein the mammal is a human.

[3] The display seal as described in [1] or [2] above, which contains 2-propanol as a component of exhaust gas.

[4] The display seal as described in any one of [1] to [3] is less outgassing.

[5] A method for culturing fertilized eggs or cells, characterized by culturing the collected mammalian fertilized eggs or mammals in a culture container attached with the display seal as described in any one of [1] to [4] above cell.

[6] The culture method as described in [5] above, wherein the mammal is a human.

[7] A kit for mammalian fertilized eggs or cell culture, characterized by comprising the display enclosure as described in any one of [1] to [4].

[8] The kit according to [7] above, wherein the mammal is human. Invention effect

Because the display seal of the present invention does not have a bad influence on the embryogenesis of the fertilized egg, the in vitro culture of the fertilized egg in the culture container attached with the display seal of the present invention can efficiently produce a good blastoderm Sac of embryo. In addition, the display seal of the present invention, which has no effect on embryos, can be expected to have no adverse effect on cell proliferation or survival even if it is used for sample identification in cell culture of ES cells, iPS cells, and nerve cells.

The sealing material for display of the present invention is limited to the use of "being attached to the surface of a culture container for mammalian fertilized eggs or cell culture", and forms a water-soluble adhesive layer on the surface of the substrate layer to reduce or inhibit the generation of outgassing For display seals, here "exhaust" refers to the gas produced by the volatilization of volatile substances. When the display seal of the present invention with such properties is used, because it can effectively reduce or suppress the amount of exhaust gas dissolved in the culture solution, it can prevent the exhaust gas released from the seal from becoming the main cause, which will affect the embryos of mammalian fertilized eggs. Occurrence or the proliferation of mammalian cells has a bad influence.

In addition, as the method for culturing the fertilized egg or cell of the present invention, if it is contained in vitro, the collected mammalian fertilized egg or mammalian cell is cultured in a culture container attached with the display seal of the present invention. There are no special restrictions, but it does not include the so-called medical actions performed by doctors, such as the steps of embryogenesis and implantation of fertilized eggs (embryos) in the state of the blastocyst in the uterus of women when cultivating mammalian fertilized eggs, or selection The steps of selecting an embryo implanted in a female uterus, such as the act of discarding a normal embryo. The culture of mammalian fertilized eggs can be carried out by the microdroplet method. In addition, the culture of mammalian cells corresponds to the type of cell, and the type of culture medium, the subculture method, etc. can be appropriately selected.

In addition, as the kit of the present invention, it is limited to the use of "for mammalian fertilized eggs or cell culture", and includes the display enclosure of the present invention. Such kits generally include those generally used for such kits, and in addition to, for example, culture vessels and culture fluids, they also contain additional documents such as instructions for the display seal of the present invention.

Examples of the above-mentioned mammals include rodents such as mice, rats, hamsters, and guinea pigs, lagomorphs such as rabbits, eyes of ungulates such as pigs, cows, goats, horses, and sheep, and meats such as dogs and cats. Orders, such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees and other primates, of which humans can be better exemplified.

As the above-mentioned mammalian cells, specific examples include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), induced pluripotency Pluripotent stem cells such as stem cells (iPS cells; induced pluripotent stem cells), pluripotent stem cells such as mesenchymal stem cells, hematopoietic stem cells, nervous system stem cells, dental pulp stem cells, myocardial precursor cells, vascular endothelial precursor cells, neural precursor cells, and fat Stem cells such as monopotent stem cells (precursor cells) such as precursor cells, skin fibroblasts, skeletal muscle myoblasts, osteoblasts, and dental cells, or cardiomyocytes, vascular endothelial cells, nerve cells, fat cells, skin fiber cells , Skeletal muscle cells, bone cells, hepatocytes, umbilical vein endothelial cells, skin microlymphatic endothelial cells, epidermal keratinocytes, bronchial epithelial cells, melanocytes, smooth muscle cells, tooth cells, dermal papillary cells and other mature cells. In addition, the above-mentioned mammals also include cells derived from the seminiferous tubules of organ cultured testicles or cells derived from self-cultured skin. Furthermore, the aforementioned mammalian cells may be cells in a tissue state.

As a base material, specifically, cellophane, plastic, paper, cloth, non-woven fabric, etc. can be cited.

The thickness of the water-soluble adhesive layer is generally 5 to 250 μm, preferably 5 to 150 μm.

As components of exhaust gas, volatile substances such as formaldehyde, D-limonene, toluene, acetone, methanol, ethanol, 2-propanol, acetonitrile, 1-butanol, 2-ethylhexanol, and hexanal ( One or two or more of the organic compounds) preferably contain 2-propanol.

As the above-mentioned water-soluble adhesive, specifically, methylcellulose, sweet potato starch, potato starch, tapioca starch, wheat starch, corn starch, konjac, carrageenan, agar, sodium alginate, hibiscus, and yellow yarrow can be mentioned. Gum, acacia, polydextrose, polyfructose, gelatin, gelatin, casein, collagen, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl Cellulose, soluble starch, carboxymethyl starch, dialdehyde starch, polyvinyl alcohol, sodium polyacrylate, polyethylene oxide, polyacrylamide, polyacrylic acid, poly-N-vinylpyridine, etc.

Various non-volatile additives such as water-soluble plasticizers, surfactants, tackifiers, antioxidants, colorants, and oils can also be added to the aforementioned substrate layer or water-soluble adhesive layer.

The sealing material for display of the present invention is usually made of release paper (release film) in order to prevent adhesion of dust and the like on the surface of the water-soluble adhesive layer (single side where the substrate layer is not formed) in the unused state. protection. In order to prevent the generation of silicone gas, the sealing material for display of the present invention preferably does not contain polysiloxane (silicon-free).

As the display seal of the present invention, it is preferably one with less exhaust gas. In the present invention, "less exhaust" refers to the cultivation of the display seal of the present invention (seal sample) or multiple sheets (for example, within the range of 2-15) of the present invention In a container (a porous plate, a petri dish (dish, dish), etc.), the culture solution cultured for any period of time (usually 6 hours to 10 days, preferably 3 to 7 days) is heated (usually within the range of 40 to 150°C) , Preferably 50～70℃) The amount of gas generated at any time (usually 10～180 minutes, preferably 50～70 minutes), when the gas chromatography mass spectrometer (GC-MS) is used for detection/measurement , The peak of one or two or more of the origin components of exhaust gas is almost or completely undetected (below the detection sensitivity).

The display seal of the present invention can be produced by applying a water-soluble adhesive that reduces or suppresses the generation of exhaust on one side of a substrate that reduces or suppresses the generation of exhaust gas. In addition, as the display seal of the present invention, it is also possible to use commercially available products that are coated to reduce or suppress the generation of exhaust gas, such as the 3M (registered trademark) low exhaust adhesive transfer tape (ATX203SF or ATX204SF) manufactured by 3M. Or a silicone-free adhesive tape made by LINTEC (double-sided tape [TL-6177-12-GA], less substrate type [TL-607-GA or TL-609-GA]) or single-sided Type [H-41177-50, H-41041-50, H-41051-50 or H-41081-50].

As the display seal of the present invention, if it is the surface of a culture container (a porous plate, a petri dish (dish, dish), etc.) used for mammalian fertilized eggs or cell culture, it is better if it is used for mammalian fertilized eggs or The outer surface of the culture container that the cells contact, or when the culture container is a multi-well plate or a petri dish, it can also be the inner side of the cover of the culture container or the inner side of the cover, but it is usually not fertilized with mammalian eggs Or the outer surface of the culture container that the cells contact, or when the culture container is a multi-well plate or a petri dish, it may be the outside of the upper surface of the cover of the culture container or the outside of the peripheral surface of the cover. The number of display seals attached to the surface of the culture container is usually one. However, the display seal of the present invention has an excellent effect of not adversely affecting the embryogenesis of fertilized eggs or the proliferation/survival of mammalian cells. It can be multiple pieces corresponding to the needs (if the total area of the whole seal is within the area of the disc, for example, within the range of 2-15 pieces).

The following examples illustrate the present invention in detail, but the present invention is not limited to these examples. Example 1

1. Confirm the influence of the exhaust gas released from the display seal during embryogenesis In order to confirm the influence of the exhaust gas released from the display seal during embryogenesis, proceed in the following sequence [1]～[3] Review. 1-1 Method [1] As a water-soluble adhesive seal with less exhaust, cut the adhesive tape (thickness: 0.075mm, product number: H-41177-50, manufactured by LINTEC) to 8mm in length. , 28mm wide to use. Also, as the exhaust seal, a seal manufactured by ASONE (length: 8mm, width: 28mm, thickness: 0.07mm, product number: 72150) was used. Put these two types of seals on the inner side of the lid of the culture plate (3002, manufactured by Falcon), and 6 on the inner side of the lateral surface, and place the drops on the culture plate (1008, manufactured by Falcon). (Droplet)-shaped culture medium (M16 culture medium containing 4 mg/mL BSA [Bovine Serum Albumin]), covered with paraffin oil (OVOIL, VitroLife) (3 mL/plate), and placed on the cover removed On a culture plate (3002, manufactured by Falcon) (refer to Figure 1). In addition, as a control group, experiments were conducted using culture plates with unlabeled seals. [2] After adding mouse fertilized eggs to drop-like culture medium, culture them in an incubator (37°C, 5% O2, 5% CO2, and 90% N2). In addition, mouse fertilized eggs are produced by in vitro fertilization using 8-week-old male BDF1 and female BDF1. [3] 2 days after culture, calculate the number of fertilized eggs divided (cleavage) (refer to Table 1, Figures 2 to 4 on the second day), and on the 5th day after culture, calculate the number of fertilized eggs that have reached the blastocyst The number of fertilized eggs (refer to Table 1, Figures 2 to 4 on the 5th day), use Gardner classification to evaluate the stage of blastocyst development (grade 1; initial blastocyst [inner cavity is less than 1/2 of the whole ], grade 2; blastoderm sac [inner cavity is more than 1/2 of the whole], grade 3; complete blastoderm sac, grade 4; expanded blastoderm sac, grade 5; hatched blastoderm sac, and grade 6; hatch Post-blastoderm sac) (refer to Table 2, Day 5 of Figure 2 to Figure 4).

1-2 Results First, use GC-MS analysis to confirm that the water-soluble adhesive seal (refer to "Sample 3" in Figure 5) of the exhaust gas is treated with less exhaust gas, that is, in the water-soluble The exhaust volume of the adhesive seal (refer to "Sample 2" in Figure 5) is below the detection sensitivity. In the case of using a culture plate with a vented seal to culture fertilized eggs, the ratio of divided fertilized eggs is as low as 9% compared with the case of a culture plate without a seal, and it does not reach the blastoderm sac at all Fertilized eggs (refer to Table 1). On the other hand, in the case of culture using a culture dish with a water-soluble adhesive seal attached with less air-exhaustion, it was confirmed that the fertilized egg was completely divided and reached the blastocyst sac. The egg ratio is as high as 90%, and it is almost the standard grade 3 (complete blastoderm sac) as a good embryo (refer to Table 1). The above results indicate that the water-soluble adhesive seal that reduces or inhibits the production of volatile substances does not have a bad influence on the embryonic development of fertilized eggs, so it can be used as a culture for attaching to the culture of human fertilized eggs The display on the surface of the container is used with a seal.

表中的「*」表示相對於控制組在統計上有顯著差異（p＜0.01）。Table 1

The "*" in the table indicates that there is a statistically significant difference from the control group (p<0.01).

實施例2Table 2

Example 2

2. Identification of exhaust that has a bad influence on embryogenesis. When a vented seal is attached to the culture plate, the volatile substances released from the seal are dissolved in the culture medium, which is considered to have a bad influence on the embryogenesis of the fertilized egg. Therefore, in order to confirm the volatile substances that have a bad influence on embryonic development, the following methods are reviewed in the order of [1] to [3]. 2-1 Method [1] Put the two types of seals used in Example 1 (water-soluble adhesive seals with less exhaust gas and seals with exhaust gas) on the cover of the culture plate (3002, manufactured by Falcon) 7 pieces on the inside and 6 pieces on the inside of the lateral surface. A drop-like culture medium (M16 culture medium containing 4mg/mL BSA [Bovine Serum Albumin]) is placed on a culture plate (1008, manufactured by Falcon) , Cover it with paraffin oil (OVOIL, manufactured by VitroLife) (3mL/plate), and place it on the culture plate (3002, manufactured by Falcon) with the lid removed (refer to Figure 1). In addition, as a control group, experiments were conducted using culture plates with unlabeled seals. [2] After culturing in an incubator (37°C, 5% O ₂ , 5% CO ₂ , 90% N ₂ ) for 5 days, the culture fluid sample was recovered to a headspace vial. [3] After heating the culture medium sample at 60°C for 1 hour, it was analyzed by GC-MS using a measuring device (GCMS-TQ8030, manufactured by Shimadzu Corporation) and a column (InertCap 1, manufactured by GL Science). In addition, as a control group, analyze the heat treatment of the seal (manufactured by ASONE) that vents the gas by GC-MS (refer to the "seal sample" in Table 3).

2-2 Results Compared with the case of culture using a less exhausted seal, no volatile substances other than ethanol were detected (refer to "Culture fluid sample less exhausted" in Table 3). When using an exhausted seal In the case of culture, 2-propanol was detected in addition to ethanol (refer to "Exhaust culture fluid sample" in Table 3). From this result, 2-propanol was identified as a volatile substance that has a bad influence on embryogenesis. In addition, because ethanol was also detected in the culture medium sample without the seal attached (refer to the "culture medium sample -" in Table 3), the ethanol detected in the seal with less exhaust is not released from the seal. It is considered that ethanol is mixed in the air used for sterilization and disinfection.

表中的「○」表示各種物質被檢出，「－」表示各種物質未被檢出。 實施例3table 3

"○" in the table indicates that various substances have been detected, and "-" indicates that various substances have not been detected. Example 3

3. Quantification of 2-propanol released from the seal When attaching the vented seal to the culture plate, the 2-propanol in the culture medium is below the detection sensitivity. Therefore, it is quantitatively analyzed how much 2-propanol is released from the exhaust seal itself. 3-1 Method Heat treatment of the two types of seals used in Example 1 (water-soluble adhesive seals with less exhaust gas and seals with exhaust gas) at 60°C for 1 hour, and then use the measuring device (GCMS-TQ8030 , Manufactured by Shimadzu Corporation) and column (InertCap 1, manufactured by GL SCIENCE Corporation) for GC-MS analysis.

The 2-propanol released from the vented seal is less than the detection sensitivity compared to the one detected at 0.093μL/g, and the 2-propanol released from the vented seal is less than the detection sensitivity (less than 0.08μL/g ) (Refer to Table 4).

表中的濃度表示每單位封件重量（g）的2-丙醇量（μL）。 產業利用性Table 4

The concentration in the table represents the amount of 2-propanol (μL) per unit weight (g) of the seal. Industrial availability

The present invention contributes to the treatment of infertility.

Figure 1 is a schematic view of a culture plate to which the display seal of the present invention is attached. Figure 2 shows the microscopic images of the second and fifth days after culturing mouse fertilized eggs on a culture plate without a seal. Figure 3 shows the microscope images of the second and fifth days after culturing mouse fertilized eggs on a culture plate with a vented seal. Figure 4 shows the microscopic images of the second and fifth days after culturing mouse fertilized eggs on a culture plate attached with a water-soluble adhesive seal with less gassing. Figure 5 shows the result of analyzing the total amount of exhaust gas in a water-soluble adhesive seal with low exhaust gas ("Sample 2" in the figure) with a gas chromatography mass spectrometer (GC-MS). The sample 3 in the figure represents the sample 2 before the exhaust gas reduction treatment, and the sample 1 represents the seal of the exhaust gas.

Claims (4)

A method for culturing fertilized eggs or cells, characterized by comprising: attaching a display seal that reduces or inhibits outgas generation with a water-soluble adhesive layer formed on one side of a substrate layer to a culture container On the surface, the collected mammalian fertilized eggs or mammalian cells are cultured in a culture container attached with the aforementioned display seal. The culture method as described in item 1 of the scope of patent application, wherein the mammal is human. The display seal as described in item 1 or item 2 of the scope of patent application contains 2-propanol as a component of exhaust gas. The display seal described in item 1 or item 2 of the scope of patent application is less exhausted.

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