TW201716567A - Culturing-container display sticker - Google Patents

Abstract

The present invention addresses the problem of providing a display sticker that does not adversely affect the embryonic development of fertilized eggs or the growth and survival of mammalian cells even if the sticker is attached to a surface of a culturing container used to culture the mammalian fertilized eggs or cells, and providing a fertilized-egg or cell culturing method and a culturing kit with which it is possible to create blastocyst mammalian embryos and culture mammalian cells without adversely affecting the embryonic development of fertilized eggs or the growth and survival of cells. Because a display sticker that is formed by forming a water-soluble adhesive layer on one side of a substrate layer and in which outgas generation is reduced or suppressed does not adversely affect the embryonic development of fertilized eggs or the growth and survival of mammalian cells, it is possible to efficiently create good blastocyst embryos by culturing, in vitro, fertilized eggs in a culturing container to which the water-soluble adhesive sticker is attached.

Description

Display seal for culture container

The present invention relates to a method for cultivating a fertilized egg or a cell cultured in a culture container for attaching such a display seal to a display container attached to a surface of a culture container for mammalian fertilized egg or mammalian cell culture, Or a kit for cultivating a mammalian fertilized egg or a mammalian cell having the above-described display seal.

In Japan, due to the effects of late marriage or late birth, there is a group of couples in the four groups who have infertility problems. Therefore, in addition to general infertility treatment by artificial insemination (AIH), the implementation of high infertility treatment (ART) such as in vitro fertilization (IVF) and microinsemination (ICSI) tends to increase.

Now, the sperm and the egg cultured under the conditions of the fallopian tube close to the original fertilization site are fertilized in vitro, and after the fertilized egg is produced, it can be discharged from the stage of cleavage, morula, and blastocyst, to hatching from the yolk membrane. The stage of the blastocyst is cultured. Although the embryo is transplanted to the uterus at the stage of the blastocyst, a high pregnancy rate is obtained, but the ratio of embryos occurring to the stage of the blastocyst is 40 to 50%, especially a good blastocyst, and the ratio is as low as 20 to 30% becomes a problem.

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

On the other hand, since the disc for cultivating the patient's embryo is individually managed, although the description by the stylus is used or the sample of the seal is recognized, the seal attached to the culture tray to the fertilized egg is not considered. The possibility of embryogenesis has a bad influence. Prior Technical Literature Patent Literature

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

Problem to be solved by the invention

An object of the present invention is to provide a fertilized egg or a cell culture method or a culture kit which does not cause embryogenesis of a fertilized egg or proliferation of a mammalian cell even if it is attached to a surface of a culture container for mammalian fertilized eggs or cell culture/ Survival has a bad influence on the display seal, or does not have a bad influence on the embryogenesis or cell proliferation/survival of the fertilized egg, and the embryo of the mammalian blastocyst can be produced to culture the mammalian cells. Means of solving problems

As described above, in the in vitro culture of fertilized eggs, the problem that the display seal attached to the surface of the culture vessel adversely affects the embryogenesis of the fertilized egg is not known to those skilled in the art. The inventors of the present invention found that exhaust gas (at least 2-propanol) has a bad influence on the embryogenesis of the fertilized egg, as a result of research using a venting seal and a less vented seal (water-soluble adhesive seal). And the water-soluble adhesive seal which suppresses or reduces the venting does not adversely affect the embryogenesis of the fertilized egg, so that the fertilized egg can be cultured in vitro in a culture container to which such a water-soluble adhesive seal is attached, and can be efficiently produced efficiently. The human is waiting for the embryo of the mammalian blastocyst, and the present invention has been completed.

That is, the present invention is as follows.

[1] A display seal for attaching to a surface of a culture vessel 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 suppress exhaust gas (outgas) is produced.

[2] The display seal according to [1] above, wherein the mammal is a human.

[3] The display seal of the above [1] or [2], wherein a component of the exhaust gas contains 2-propanol.

[4] The display seal according to any one of the above [1] to [3], wherein the display is less exhausted.

[5] A method for cultivating a fertilized egg or a cell, which is characterized in that the mammalian fertilized egg or mammal is cultured in a culture container to which the display seal according to any one of the above [1] to [4] is attached. cell.

[6] The culture method according to [5] above, wherein the mammal is a human.

[7] A kit for use in a mammalian fertilized egg or a cell culture, comprising the display seal according to any one of the above [1] to [4].

[8] The kit according to [7] above, wherein the mammal is a human. Effect of the invention

Since the display seal of the present invention does not adversely affect the embryogenesis of the fertilized egg, the fertilized egg is cultured in vitro in the culture container to which the display seal of the present invention is attached, and a good blastoderm can be efficiently produced. The embryo of the capsule. Further, the display seal of the present invention which does not affect the embryo can be expected to have a bad influence on cell proliferation or survival even when used for sample identification in cell culture of ES cells, iPS cells, nerve cells and the like.

The display seal of the present invention is limited to the use of "a surface for culturing a mammalian fertilized egg or a culture vessel for cell culture", forming a water-soluble adhesive layer on the surface of the substrate layer to reduce or suppress exhaust gas generation. The display seal is used herein. "Exhaust" refers to the gas generated by the volatilization of volatile substances. When the display seal of the present invention having such a property is used, since the amount of exhaust gas dissolved in the culture solution can be effectively reduced or suppressed, the exhaust gas released from the seal can be prevented from being the main cause, and the embryo of the fertilized egg of the mammal is fertilized. Occurrence or proliferation of mammalian cells has a bad influence.

In addition, a method of culturing a fertilized egg or a cell of the present invention, which is a method of culturing a mammalian fertilized egg or a mammalian cell which is taken in vitro, and culturing it in a culture container to which the display seal of the present invention is attached There is no particular limitation, but it is not included in the medical practice of the so-called physician, such as the step of implanting the fertilized egg (embryo) in the blastocyst state, in the step of implanting the female uterus, in the case of culturing a mammalian fertilized egg. The step of selecting the embryo that is placed in the female uterus, etc., has the behavior of discarding the normal embryo. The culture of mammalian fertilized eggs can be carried out by the microdroplet method. Further, the culture of the mammalian cells is performed in accordance with the type of the cell, and the type of the culture solution, the method of subculture, and the like can be appropriately selected.

Further, the kit of the present invention is limited to the "use for mammalian fertilized eggs or cell culture" and includes a kit for display closure of the present invention. In such a kit, generally, it is generally included in the kit, and includes, for example, a culture container and a culture solution, and an additional document such as a description of the display seal of the present invention.

The above-mentioned mammals can be exemplified by rodents such as mice, rats, hamsters, and guinea pigs, rabbits and other hoofed eyes such as pigs, cows, goats, horses, and sheep, such as dogs and cats. A primate such as a human, a monkey, a rhesus monkey, a cynomolgus monkey, a marmoset, an orangutan, a chimpanzee, etc., which may preferably be exemplified as a human.

As the above mammalian cells, specifically, embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), and induced pluripotency can be cited. 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 stem cells, dental pulp stem cells, myocardial precursor cells, vascular endothelial precursor cells, neural precursor cells, and fat Stem cells such as precursor cells, dermal fibroblasts, skeletal muscle myoblasts, osteoblasts, and dendritic cells, such as single-potential stem cells (precursor cells), or cardiomyocytes, vascular endothelial cells, nerve cells, fat cells, and skin fibroblasts Mature cells such as skeletal muscle cells, bone cells, liver cells, umbilical vein endothelial cells, skin microlymphatic endothelial cells, epidermal keratinocytes, bronchial epithelial cells, melanocytes, smooth muscle cells, tooth cells, and dermal papilla cells. Further, in the mammal, the cells derived from the seminiferous tubules of the organ cultured pellets or the cells derived from the self-cultivating skin are also included. Furthermore, the above mammalian cells may also be cells in a tissue state.

Specific examples of the substrate include cellophane, plastic, paper, cloth, and non-woven fabric.

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

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

As the above water-soluble adhesive, specifically, methyl cellulose, sweet potato starch, potato starch, tapioca starch, wheat starch, corn starch, alfalfa, carrageen, amaranth, sodium alginate, hibiscus, and scutellaria are mentioned. Gum, gum arabic, 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, polypropylene decylamine, polyacrylic acid, poly-N-vinyl pyridine, and the like.

Various non-volatile additives such as water-soluble plasticizers, surfactants, tackifiers, antioxidants, colorants, and oils may be added to the base material layer or the water-soluble adhesive layer.

The display seal of the present invention usually has a release paper (release film) in order to prevent dust or the like from adhering to the surface of the water-soluble adhesive layer in an unused state (a single surface of the base material layer is not formed). protection. Further, in order to prevent the generation of the deuterium gas, the display seal of the present invention preferably does not contain polyfluorene (no antimony).

As the display seal of the present invention, it is preferable to have less exhaust. In the present invention, "less exhaust gas" means the culture of the display seal of the present invention attached to the display seal (package sample) or a plurality of sheets (for example, in the range of 2 to 15 sheets) of the present invention. In a container (porous disk, petri dish (disc, dish), etc.), the culture medium cultured at any time (usually 6 hours to 10 days, preferably 3 to 7 days) is heated (usually in the range of 40 to 150 ° C). The amount of gas generated at any time (usually 10 to 180 minutes, preferably 50 to 70 minutes) at a temperature of 50 to 70 ° C is detected/measured by gas chromatography mass spectrometry (GC-MS). The peak of the origin component of one or two or more kinds of exhaust gas is a state in which it is almost or completely undetected (detection sensitivity or less).

The display seal of the present invention can be produced by coating or reducing the water-soluble adhesive generated by the exhaust gas on one side of the substrate which reduces or suppresses the generation of exhaust gas. Further, as the display seal of the present invention, a commercially available product which reduces or suppresses the generation of exhaust gas, for example, a 3M (registered trademark) low-exhaust adhesive transfer tape (ATX203SF or ATX204SF) manufactured by 3M Company, may be used. Or adhesive tape made of LINTEC, which is flawless and rarely vented (double-sided tape [TL-6177-12-GA], less substrate type [TL-607-GA or TL-609-GA]) or single-sided adhesive Type [H-41177-50, H-41041-50, H-41051-50 or H-41081-50].

As a display seal to which the present invention is attached, it is preferably a surface of a culture container (porous plate, petri dish (disc, dish), etc.) for mammalian fertilized eggs or cell culture, and is fertilized with mammalian eggs or The outer surface of the culture vessel in contact with the cells, or in the case where the culture vessel is a porous disk or a petri dish, may be the inner side of the upper surface of the lid of the culture vessel or the inner side of the lid side, but usually not with the mammalian fertilized egg Alternatively, when the culture container is a porous disk or a petri dish, or the culture container is a porous disk or a petri dish, the outer side of the lid of the culture container or the outer side of the lid side peripheral surface may be used. The number of sheets of the display seal attached to the surface of the culture container is usually one piece, but the display seal of the present invention has a superior effect of not adversely affecting the embryogenesis of the fertilized egg or the proliferation/survival of the mammalian cells. It may be a plurality of pieces corresponding to the need (if the total area of the full seal is within the area of the disk, for example, in the range of 2 to 15 pieces).

The invention is described in detail below by way of examples, but the invention is not limited thereto. Example 1

1. Confirmation of the influence of the exhaust gas released from the display seal in the embryo to confirm the influence of the exhaust gas released from the display seal in the embryo, according to the following methods [1] to [3] Review. 1-1 Method [1] As a water-soluble adhesive seal with less exhaust gas, the adhesive tape (thickness: 0.075 mm, product number: H-41177-50, manufactured by LINTEC) with very little exhaust gas is cut and grown to 8 mm. , 28mm wide to use. Further, as a seal for the exhaust, a seal manufactured by ASONE (length: 8 mm, width: 28 mm, thickness: 0.07 mm, product number: 72150) was used. These two types of seals were attached to the inside of the cover of the culture tray (3002, manufactured by falcon), and 6 sheets were placed on the inner side of the side peripheral surface, and the drop was placed on the culture tray (1008, manufactured by Falcon). (droplet) culture solution (M16 culture solution containing 4 mg/mL BSA [bovine serum albumin]), which was covered with paraffin oil (OVOIL, manufactured by VitroLife) (3 mL/plate), and placed in a cap removal The culture plate (3002, manufactured by Falcon Co., Ltd.) was used (see Fig. 1). Further, as a control group, an experiment using a culture plate without a label seal was performed. [2] After the mouse fertilized egg was added to the dropping culture solution, it was cultured in an incubator (37 ° C, 5% O 2 , 5% CO 2 , 90% N 2 ). Further, the mouse fertilized egg was prepared by in vitro fertilization using 8-week-old male BDF1 and female BDF1 according to a conventional method. [3] Two days after the culture, the number of fertilized eggs that were divided (cleavage) was calculated (see Table 2, Day 2 to Figure 2 on the second day), and on the fifth day after the culture, the arrival of the blastocysts was calculated. The number of fertilized eggs (see Table 5, Day 5 to Figure 4 on the 5th day), the Gardner classification was used to evaluate the stage of blastocyst (grade 1; initial blastocysts [the lumen is less than 1/2 of the whole) ], grade 2; blastoderm [inner lumen is more than 1/2 of the whole], grade 3; complete blastocyst, grade 4; dilated blastocyst, grade 5; hatching blastocyst, and grade 6; hatching Posterior blastocyst) (see Table 5, Day 5 to Figure 4, Day 5).

1-2 Results First, it was confirmed by GC-MS analysis that the water-soluble adhesive seal of the exhaust gas (see "Sample 3" in Fig. 5) was treated with less exhaust gas, that is, water dissolved in less exhaust gas. The amount of exhaust of the adhesive seal (refer to "Sample 2" in Fig. 5) is the detection sensitivity or less. The cultured dish with the vented seal is used to culture the fertilized egg. Compared with the case of the culture plate without the seal, the ratio of the fertilized egg is as low as 9%, and the blastocyst is not reached at all. Fertilized eggs (see Table 1). On the other hand, in the case of culturing using a culture plate to which a water-soluble adhesive seal having a small amount of exhaust gas is attached, as in the case of a culture plate to which a seal is not attached, it is confirmed that the fertilized egg is completely divided and the fertilization is reached to the blastocyst. The egg ratio is as high as 90%, and is almost the blastocyst sac above the standard grade 3 (complete blastoderm) of good embryos (see Table 1). The above results indicate that the water-soluble adhesive seal which reduces or inhibits the generation of volatile substances does not adversely affect the embryogenesis of the fertilized egg, and therefore can be used as a culture for attaching to a cultured fertilized egg. The display of the surface of the container is used with a seal.

Table 1 The "*" in the table indicates a statistically significant difference (p < 0.01) with respect to the control group.

Table 2 Example 2

2. Identification of exhaust gas that has a bad influence on embryogenesis When the seal attached to the exhaust gas is placed on the culture plate, the volatile matter released from the seal is dissolved in the culture solution, and it 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 embryogenesis, the review is carried out in the following order [1] to [3]. 2-1 Method [1] The two types of seals used in Example 1 (water-soluble adhesive seals and exhaust seals with less exhaust gas) were placed on the cover of a culture tray (3002, manufactured by Falcon). 7 pieces were attached to the inside, and 6 pieces were attached to the inside of the side peripheral surface, and a dropping medium (M16 medium containing 4 mg/mL BSA [bovine serum albumin]) was placed on a culture plate (1008, manufactured by Falcon Co., Ltd.). It was covered with paraffin oil (OVOIL, manufactured by VitroLife) (3 mL/disc), and placed on a culture tray (3002, manufactured by Falcon Co., Ltd.) from which the cap was removed (see Fig. 1). Further, as a control group, an experiment using a culture plate without a label seal was performed. [2] After incubating for 5 days in an incubator (37 ° C, 5% O ₂ , 5% CO ₂ , 90% N ₂ ), the culture solution sample was recovered to a headspace vial. [3] The culture solution sample was heat-treated at 60 ° C for 1 hour, and then 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, the seal of the exhaust gas (manufactured by ASONE) itself was analyzed by GC-MS (see "Package Sample" of Table 3).

2-2. In the case of culturing with a seal that uses less exhaust gas, volatile substances other than ethanol are not detected (see "Cultivating liquid sample less exhaust" in Table 3), and seals using exhaust gas are used. In the case of culture, 2-propanol was detected in addition to ethanol (refer to "Extraction of culture solution sample" in Table 3). From this result, it was identified that a volatile substance which has a bad influence on embryogenesis is 2-propanol. In addition, since ethanol is also detected in the culture liquid sample to which the seal is not attached (refer to "culture liquid sample -" in Table 3), the ethanol detected in the seal with less exhaust gas is not released from the seal. It is considered to be an admixture of ethanol in the air used for sterilization and disinfection.

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

3. Quantification of 2-propanol released from the seal When the seal attached to the exhaust is placed on the culture tray, 2-propanol in the culture solution is below the detection sensitivity. Therefore, quantitative analysis of how much 2-propanol is released about the seal of the exhaust gas itself. 3-1 Method The two types of seals used in Example 1 (water-soluble adhesive seals with less exhaust gas and exhaust seals) were heat-treated at 60 ° C for 1 hour, by using a measuring device (GCMS-TQ8030). , manufactured by Shimadzu Corporation) and pipe column (InertCap 1, manufactured by GL SCIENCE) by GC-MS.

2-propanol released from the seal of the exhaust gas, the 2-propanol released from the seal of the exhaust gas is below the detection sensitivity (not more than 0.08 μL/g) with respect to 0.093 μL/g. ) (Refer to Table 4).

Table 4 The concentration in the table indicates the amount of 2-propanol (μL) per unit of seal weight (g). Industrial utilization

The present invention is contributing to infertility treatment.

Fig. 1 is a schematic view of a culture tray to which a display seal of the present invention is attached. Fig. 2 is a view showing a microscope image on the second and fifth days after the mouse fertilized eggs were cultured using a culture plate to which the seal was attached. Fig. 3 is a view showing a microscope image on the second and fifth days after culturing a mouse fertilized egg with a culture plate to which a ventilated seal is attached. Fig. 4 is a view showing a microscope image on the second and fifth days after culturing a mouse fertilized egg with a culture plate in which a water-soluble adhesive seal having less exhaust gas is attached. Fig. 5 is a graph showing the results of analyzing the total amount of exhaust gas in a water-soluble adhesive seal ("sample 2" in the drawing) with less exhaust gas by gas chromatography mass spectrometry (GC-MS). Sample 3 in the figure indicates sample 2 before the exhaust treatment, and sample 1 indicates the seal of the exhaust.

Claims (8)

A display seal for attaching to a surface of a culture vessel 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 suppress outgas produce. The display seal of claim 1, wherein the mammal is a human. The display seal of claim 1 or 2, wherein a component of the exhaust gas comprises 2-propanol. The display seal according to any one of claims 1 to 3 is less exhausted. A method for cultivating a fertilized egg or a cell, which is characterized in that a mammalian fertilized egg or mammal is taken from a culture container to which a display seal according to any one of claims 1 to 4 is attached. cell. The culture method according to claim 5, wherein the mammal is a human. A kit for use in a mammalian fertilized egg or a cell culture, comprising the display seal according to any one of claims 1 to 4. The kit of claim 7, wherein the mammal is a human.