JP6774420B2 - Labeling sticker for culture container - Google Patents
Labeling sticker for culture container Download PDFInfo
- Publication number
- JP6774420B2 JP6774420B2 JP2017544183A JP2017544183A JP6774420B2 JP 6774420 B2 JP6774420 B2 JP 6774420B2 JP 2017544183 A JP2017544183 A JP 2017544183A JP 2017544183 A JP2017544183 A JP 2017544183A JP 6774420 B2 JP6774420 B2 JP 6774420B2
- Authority
- JP
- Japan
- Prior art keywords
- seal
- culture
- cells
- outgas
- fertilized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
本発明は、哺乳類受精卵又は哺乳類細胞の培養に用いる培養容器表面に貼付するための表示用シールや、かかる表示用シールを貼付した培養容器中で培養する哺乳類受精卵又は哺乳類細胞の培養方法や、上記表示用シールを備えた、哺乳類受精卵又は哺乳類細胞の培養用キットに関する。
INDUSTRIAL APPLICABILITY The present invention provides a labeling sticker for affixing to the surface of a culture vessel used for culturing fertilized mammalian eggs or mammalian cells, a method for culturing fertilized mammalian eggs or mammalian cells to be cultured in a culture vessel to which such a labeling sticker is affixed. The present invention relates to a kit for culturing fertilized mammalian eggs or mammalian cells, which comprises the above-mentioned display seal.
日本においては、晩婚化や晩産化の影響により、４組に１組の夫婦が不妊の問題を抱えているといわれている。このため、人工授精（ＡＩＨ）による一般的な不妊治療の他、体外受精（ＩＶＦ）、顕微授精（ＩＣＳＩ）等の高度不妊治療（ＡＲＴ）の実施が増加傾向にある。 In Japan, it is said that one in four couples has a problem of infertility due to the effects of late marriage and late birth. Therefore, in addition to general infertility treatment by artificial insemination (AIH), implementation of advanced infertility treatment (ART) such as in vitro fertilization (IVF) and microinsemination (ICSI) is increasing.
現在、本来の受精の場である卵管に近い条件下で培養した精子と卵子とを体外受精させ、受精卵を作製した後、卵割、桑実胚、胚盤胞の段階を経て、透明帯から孵化した脱出胚盤胞の段階まで培養することが可能となっている。胚盤胞の段階で胚を子宮に移植すると、より高い妊娠率が得られるが、胚盤胞の段階まで発生が進行する胚の割合は４０〜５０％であり、良好な胚盤胞に限ってはその割合が２０〜３０％と低いことが問題とされていた。 Currently, sperms and eggs cultured under conditions close to the original fertilization site, the oviduct, are fertilized in vitro to produce fertilized eggs, which are then transparent through the cleavage, mulberry embryo, and blastocyst stages. It is possible to culture from the zone to the stage of the prolapsed blastocyst that has hatched. Transplanting embryos into the uterus at the blastocyst stage results in higher pregnancy rates, but the proportion of embryos that progress to the blastocyst stage is 40-50%, limited to good blastocysts. The problem was that the ratio was as low as 20 to 30%.
受精卵の生体外培養は、通常、シャーレ上に培養液の２０〜３０μＬのドロップ（滴）を形成させ、かかるドロップ内に単一又は複数の受精卵を入れて培養を行う方法（マイクロドロップレット法）により行われる。かかるマイクロドロップレット法を改良するために、ドロップ内の受精卵を、ピペットによる操作性が向上し、かつ安定な培養液のドロップを形成できる培養容器が報告されている（特許文献１）。また、受精卵と子宮内膜細胞とを共培養させることにより、より生体内に近い環境で培養させ、発生効率を向上させる方法も報告されている（特許文献２）。
一方、患者の胚を培養するシャーレは、個別管理されるため、マジックでの記載やシールによるサンプルの識別が行われているが、これまで培養シャーレに貼付するシールが、受精卵の胚発生に悪影響を及ぼす可能性については考えられていなかった。In vitro culture of fertilized eggs is usually performed by forming 20 to 30 μL drops of the culture solution on a petri dish and placing one or more fertilized eggs in the drops (microdroplets). It is done by law). In order to improve the microdroplet method, a culture vessel has been reported in which the fertilized egg in the drop can be easily operated by a pipette and can form a stable drop of the culture solution (Patent Document 1). In addition, a method of co-culturing fertilized eggs and endometrial cells to culture them in an environment closer to that of a living body and improving developmental efficiency has also been reported (Patent Document 2).
On the other hand, since the petri dish for culturing the patient's embryo is individually managed, the sample is identified by the description with magic and the seal, but until now, the seal attached to the cultured petri dish has been used for embryonic development of fertilized eggs. No consideration was given to the potential for adverse effects.
本発明の課題は、哺乳類受精卵又は細胞の培養に用いる培養容器表面に貼付しても、受精卵の胚発生や哺乳類細胞の増殖・生存に悪影響を及ぼすことのない表示用シールや、受精卵の胚発生や細胞の増殖・生存に悪影響を及ぼすことなく、哺乳類胚盤胞の胚を作製したり、哺乳細胞の培養を行うことができる、受精卵又は細胞の培養方法や培養用キットを提供することにある。 The subject of the present invention is a display sticker and a fertilized egg that do not adversely affect the embryonic development of the fertilized egg or the proliferation and survival of the mammalian cell even when attached to the surface of the fertilized mammalian egg or the culture vessel used for culturing the cell. Provided are fertilized egg or cell culturing methods and culturing kits capable of producing mammalian blastocyst embryos and culturing mammalian cells without adversely affecting embryonic development and cell proliferation / survival. To do.
前述のとおり、受精卵の体外培養において、培養容器表面に貼付した表示用シールが、受精卵の胚発生に悪影響を及ぼすという課題自体、この分野の当業者に知られていなかった。本発明者らは、アウトガスのシールと、アウトガスレスのシール（水溶性粘着シール）とを用いて検討した結果、アウトガス（少なくとも２−プロパノール）が受精卵の胚発生に悪影響を及ぼすことや、アウトガスが低減又は抑制された水溶性粘着シールは、受精卵の胚発生に悪影響を及ぼすことがないため、かかる水溶性粘着シールを貼付した培養容器中で受精卵を体外培養すると、効率よく良好なヒト等の哺乳類胚盤胞の胚を作製できることを見いだし、本発明の完成するに至った。 As described above, in the in vitro culture of a fertilized egg, the problem that the display sticker attached to the surface of the culture vessel adversely affects the embryonic development of the fertilized egg has not been known to those skilled in the art. As a result of studies using an outgas seal and an outgasless seal (water-soluble adhesive seal), the present inventors have found that outgas (at least 2-propanol) adversely affects embryogenesis of fertilized eggs and that outgas Since the water-soluble adhesive seal in which the amount is reduced or suppressed does not adversely affect the embryonic development of the fertilized egg, in vitro culture of the fertilized egg in the culture vessel to which the water-soluble adhesive seal is attached is efficient and good for humans. We have found that embryos of mammalian blastocysts can be produced, and have completed the present invention.
すなわち、本発明は以下のとおりである。
［１］基材層の片面に水溶性粘着剤層が形成されてなり、アウトガスの発生が低減又は抑制されていることを特徴とする、哺乳類受精卵又は哺乳類細胞の培養に用いる培養容器表面に貼付するための表示用シール。
［２］哺乳類がヒトであることを特徴とする上記［１］に記載の表示用シール。
［３］アウトガスの成分の１つとして、２−プロパノールが含まれることを特徴とする上記［１］又は［２］に記載の表示用シール。
［４］アウトガスレスであることを特徴とする上記［１］〜［３］のいずれかに記載の表示用シール。
［５］採取された哺乳類受精卵、又は哺乳類細胞を、上記［１］〜［４］のいずれかに記載の表示用シールを貼付した培養容器中で培養することを特徴とする哺乳類受精卵又は哺乳類細胞の培養方法。
［６］哺乳類がヒトであることを特徴とする上記［５］に記載の培養方法。
［７］上記［１］〜［４］のいずれかに記載の表示用シールを備えたことを特徴とする、哺乳類受精卵又は哺乳類細胞の培養に用いるためのキット。
［８］哺乳類がヒトであることを特徴とする上記［７］に記載のキット。
That is, the present invention is as follows.
[1] On the surface of a culture vessel used for culturing fertilized mammalian eggs or mammalian cells, which is characterized in that a water-soluble pressure-sensitive adhesive layer is formed on one side of a base material layer to reduce or suppress the generation of outgas. Display sticker for sticking.
[2] The display sticker according to the above [1], wherein the mammal is a human.
[3] The display sticker according to the above [1] or [2], which contains 2-propanol as one of the components of the outgas.
[4] The display sticker according to any one of the above [1] to [3], which is characterized by being outgassed.
[5] The harvested mammalian fertilized eggs, or mammalian cells, the above-mentioned [1] to [4] Mammalian fertilized eggs or characterized by culturing in a culture vessel was attached to display seal according to any one of Method of culturing mammalian cells.
[6] The culture method according to the above [5], wherein the mammal is a human.
[7] A kit for use in culturing a fertilized mammalian egg or a mammalian cell, which comprises the display sticker according to any one of the above [1] to [4].
[8] The kit according to the above [7], wherein the mammal is a human.
本発明の表示用シールは、受精卵の胚発生に悪影響を及ぼすことがないため、本発明の表示用シールを貼付した培養容器中で受精卵を体外培養すると、効率よく良好な哺乳類胚盤胞の胚を作製することができる。また、胚への影響がない本発明の表示用シールは、ＥＳ細胞、ｉＰＳ細胞、神経細胞などの細胞培養中のサンプルの識別に使用されても、細胞増殖や生存に悪影響がないことが期待される。 Since the display seal of the present invention does not adversely affect the embryonic development of the fertilized egg, when the fertilized egg is in vitro cultured in the culture vessel to which the display seal of the present invention is attached, it is efficient and good mammalian blastocyst. Embryos can be produced. In addition, the display seal of the present invention, which has no effect on embryos, is expected to have no adverse effect on cell proliferation and survival even when used for identifying samples in cell culture such as ES cells, iPS cells, and nerve cells. Will be done.
本発明の表示用シールは、「哺乳類受精卵又は哺乳類細胞の培養に用いる培養容器表面に貼付するため」という用途が限定された、基材層の片面に水溶性粘着剤層が形成されてなり、アウトガスの発生が低減又は抑制されている表示用シールであり、ここで「アウトガス」とは、揮発性物質が揮発することにより発生するガスを意味する。かかる性質を有する本発明の表示用シールを用いると、培養液中へ溶解するアウトガスの量を効果的に低減又は抑制できるため、シールから放出されるアウトガスが要因となり、哺乳類受精卵の胚発生や、哺乳類細胞の増殖に悪影響を及ぼすことを予防できる。
The display sticker of the present invention has a water-soluble pressure-sensitive adhesive layer formed on one side of a base material layer for which the use is limited to "to be attached to the surface of a culture vessel used for culturing fertilized mammalian eggs or mammalian cells". , A display sticker in which the generation of outgas is reduced or suppressed, and here, "outgas" means a gas generated by volatilization of a volatile substance. When the display seal of the present invention having such properties is used, the amount of outgas dissolved in the culture solution can be effectively reduced or suppressed. Therefore, the outgas released from the seal becomes a factor, and embryonic development of fertilized mammalian eggs or , Can prevent adverse effects on mammalian cell proliferation.
また、本発明の受精卵又は細胞の培養方法としては、インビトロで、採取された哺乳類受精卵、又は哺乳類細胞を、本発明の表示用シールを貼付した培養容器中で培養する工程を含む方法であれば特に制限されないが、哺乳類受精卵を培養する場合、胚発生が進行し、胚盤胞の状態になった受精卵（胚）を女性の子宮に着床させる工程等のいわゆる医師による医療行為や、女性の子宮に着床させる胚を取捨選択する工程等の正常な胚を破棄するおそれのある行為は含まれない。哺乳類受精卵の培養は、マイクロドロップレット法により行うことができる。また、哺乳類細胞の培養は、細胞の種類に応じて、培養液の種類、継代方法等を適宜選択して行うことができる。 In addition, the method for culturing the fertilized egg or cell of the present invention includes a step of culturing the collected mammalian fertilized egg or the mammalian cell in a culture vessel to which the display sticker of the present invention is affixed in vitro. If there is no particular limitation, but when culturing fertilized mammalian eggs, so-called medical practice by a doctor, such as the process of implanting a fertilized egg (embryo) in the state of a blastocyst in the uterus of a woman as embryonic development progresses. It does not include actions that may destroy normal embryos, such as the process of selecting embryos to implant in the womb of a woman. Fertilized mammalian eggs can be cultured by the microdroplet method. In addition, mammalian cells can be cultured by appropriately selecting the type of culture medium, the subculture method, and the like according to the type of cells.
また、本発明のキットとしては、「哺乳類受精卵又は哺乳類細胞の培養に用いるため」という用途が限定された、本発明の表示用シールを備えたキットである。かかるキットには、一般にこの種のキットに用いられるもの、例えば、培養容器、培養液の他、本発明の表示用シールについての説明書等の添付文書が通常含まれる。 In addition, the kit of the present invention is a kit provided with a display sticker of the present invention for which the use "for use in culturing fertilized mammalian eggs or mammalian cells" is limited. Such kits typically include those commonly used in this type of kit, such as culture vessels, culture solutions, as well as package inserts such as instructions for labeling stickers of the present invention.
上記哺乳類としては、マウス、ラット、ハムスター、モルモット等のげっ歯類、ウサギ等のウサギ目、ブタ、ウシ、ヤギ、ウマ、ヒツジ等の有蹄目、イヌ、ネコ等のネコ目、ヒト、サル、アカゲザル、カニクイザル、マーモセット、オランウータン、チンパンジーなどの霊長類等を例示することができ、中でもヒトを好適に例示することができる。 The mammals include rodents such as mice, rats, hamsters and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, horses and sheep, cats such as dogs and cats, humans and monkeys. , Primates such as red-tailed monkeys, cynomolgus monkeys, marmosets, orangutans, orangutans, and the like, and among them, humans can be preferably exemplified.
上記哺乳類細胞としては、具体的に胚性幹細胞（embryonic stem cells：ＥＳ細胞）、胚性生殖細胞（embryonic germ cells：ＥＧ細胞）、生殖細胞系列幹細胞（germline stem cells：ＧＳ細胞）、誘導多能性幹細胞（ｉＰＳ細胞；induced pluripotent stem cell）等の多能性幹細胞、間葉系幹細胞、造血系幹細胞、神経系幹細胞、歯髄幹細胞等の複能性幹細胞、心筋前駆細胞、血管内皮前駆細胞、神経前駆細胞、脂肪前駆細胞、皮膚線維芽細胞、骨格筋筋芽細胞、骨芽細胞、象牙芽細胞等の単能性幹細胞（前駆細胞）などの幹細胞や、心筋細胞、血管内皮細胞、神経細胞、脂肪細胞、皮膚線維細胞、骨格筋細胞、骨細胞、ヘパトサイト（肝）細胞、臍帯静脈内皮細胞、皮膚微小リンパ管内皮細胞、表皮角化細胞、気管支上皮細胞、メラノサイト細胞、平滑筋細胞、象牙細胞、毛乳頭細胞等の成熟細胞を挙げることができる。また、上記哺乳類細胞には、器官培養する精巣の精細菅由来の細胞や、自家培養皮膚由来の細胞も含まれる。なお、上記哺乳類細胞は、組織の状態の細胞であってもよい。 Specific examples of the mammalian cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), germline stem cells (GS cells), and inducible pluripotency. Pluripotent stem cells such as induced pluripotent stem cells (iPS cells), pluripotent stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, dental pulp stem cells, myocardial precursor cells, vascular endothelial precursor cells, nerves Stem cells such as progenitor cells, adipose progenitor cells, cutaneous fibroblasts, skeletal myoblasts, osteoblasts, denticle blasts and other unipotent stem cells (precursor cells), myocardial cells, vascular endothelial cells, nerve cells, Fat cells, skin fibrous cells, skeletal muscle cells, bone cells, hepatite (liver) cells, umbilical vein endothelial cells, cutaneous microlymphatic vessel endothelial cells, epidermal keratinized cells, bronchial epithelial cells, melanosite cells, smooth muscle cells, ivory cells , Mature cells such as dermal papilla cells. The mammalian cells also include cells derived from the seminiferous tubules of the testis for organ culture and cells derived from autologous skin. The mammalian cell may be a cell in a tissue state.
上記基材としては、具体的に、セロハン、プラスチック、紙、布、不織布等を挙げることができる。 Specific examples of the base material include cellophane, plastic, paper, cloth, and non-woven fabric.
上記水溶性粘着剤層の厚さは、通常５〜２５０μｍであり、好ましくは５〜１５０μｍである。 The thickness of the water-soluble pressure-sensitive adhesive layer is usually 5 to 250 μm, preferably 5 to 150 μm.
上記アウトガスの成分として、ホルムアルデヒド、Ｄ−リモネン、トルエン、アセトン、メタノール、エタノール、２−プロパノール、アセトニトリル、１−ブタノール、２−エチルヘキサノール、ヘキサナール等の揮発性物質（有機化合物）の１又は２種以上を挙げることができ、好ましくは２−プロパノールが含まれる。 As a component of the outgas, one or two kinds of volatile substances (organic compounds) such as formaldehyde, D-limonene, toluene, acetone, methanol, ethanol, 2-propanol, acetonitrile, 1-butanol, 2-ethylhexanol, and hexanal. The above can be mentioned, and 2-propanol is preferably included.
上記水溶性粘着剤としては、具体的に、メチルセルロース、かんしょ澱粉、ばれいしょ澱粉、タピオカ澱粉、小麦澱粉、コーンスターチ、こんにゃく、ふのり、寒天、アルギン酸ナトリウム、トロロアオイ、トラガントゴム、アラビアゴム、デキストラン、レバン、にかわ、ゼラチン、カゼイン、コラーゲン、ビスコース、エチルセルロース、ヒドロキシエチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロース、可溶性澱粉、カルボキシメチル澱粉、ジアルデヒド澱粉、ポリビニルアルコール、ポリアクリル酸ナトリウム、ポリエチレンオキシド、ポリアクリルアミド、ポリアクリル酸、ポリ−Ｎ−ビニルピドリドン等を挙げることができる。 Specific examples of the water-soluble pressure-sensitive adhesive include methyl cellulose, citrus starch, potato starch, tapioca starch, wheat starch, corn starch, konjac, furori, agar, sodium alginate, trolley aoi, tragant rubber, arabic rubber, dextran, levan, and sardine. Gelatin, casein, collagen, biscous, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl starch, soluble starch, carboxymethyl starch, dialdehyde starch, polyvinyl alcohol, sodium polyacrylate, polyethylene oxide, polyacrylamide, Examples thereof include polyacrylic acid and poly-N-vinylpidridone.
上記基材層や水溶性粘着剤層には、不揮発性の各種添加剤、例えば水溶性可塑剤、界面活性剤、粘着付与剤、酸化防止剤、着色剤、油剤を添加してもよい。 Various non-volatile additives such as water-soluble plasticizers, surfactants, tackifiers, antioxidants, colorants, and oils may be added to the base material layer and the water-soluble pressure-sensitive adhesive layer.
本発明の表示用シールは、未使用の状態で水溶性粘着剤層表面（基材層が形成されていない片面）に、ゴミ等が付着せずに粘着性能が維持できるように、通常、剥離紙（剥離フィルム）で保護されている。また、本発明の表示用シールは、シロキサンガスの発生を防止するために、シリコーンを含まない（シリコーンフリー）のものが好ましい。 The display sticker of the present invention is usually peeled off so that the adhesive performance can be maintained without dust or the like adhering to the surface of the water-soluble adhesive layer (one side on which the base material layer is not formed) in an unused state. It is protected by paper (release film). Further, the display seal of the present invention preferably contains no silicone (silicone-free) in order to prevent the generation of siloxane gas.
本発明の表示用シールとしては、アウトガスレスであることが好ましい。本発明において「アウトガスレス」とは、本発明の表示用シール（シールサンプル）、又は、本発明の表示用シールを複数枚（例えば２〜１５枚の範囲内）貼付した培養容器（マルチウエルプレート、培養皿［シャーレ、ディッシュ］等）中で任意の時間（通常６時間〜１０日間、好ましくは３〜７日間）インキュベートした培養液（培養液サンプル）を、任意の時間（通常１０〜１８０分間、好ましくは５０分〜７０分間）加熱（通常４０〜１５０℃の範囲内、好ましくは５０〜７０℃）したときに発生するガス量を、ガスクロマトグラフ質量分析計（ＧＣ−ＭＳ）で検出・定量した場合、１又は２種以上のアウトガスの成分由来のピークが、ほとんど又は全く検出されないこと（検出感度以下であること）を意味する。 The display seal of the present invention is preferably outgasless. In the present invention, "outgasless" means a culture container (multi-well plate) to which a display sticker (seal sample) of the present invention or a plurality of display stickers of the present invention (for example, within the range of 2 to 15) are attached. , Culture dish [Petri dish, dish], etc.) for any time (usually 6 to 10 days, preferably 3 to 7 days), and the culture solution (culture solution sample) was incubated for any time (usually 10 to 180 minutes). The amount of gas generated when heated (usually in the range of 40 to 150 ° C., preferably 50 to 70 ° C.) is detected and quantified by a gas chromatograph mass spectrometer (GC-MS). If this is the case, it means that peaks derived from one or more kinds of outgas components are hardly detected (or less than the detection sensitivity).
本発明の表示用シールは、アウトガスの発生が低減又は抑制されている基材に片面に、アウトガスの発生が低減又は抑制されている水溶性粘着剤を塗工することにより作製することができる。また、本発明の表示用シールとしては、アウトガスの発生が低減又は抑制されている市販品、例えば、３Ｍ社製の３Ｍ（登録商標）低アウトガス接着剤転写テープ（ＡＴＸ２０３ＳＦ、又はＡＴＸ２０４ＳＦ）や、LINTEC社製のシリコーンフリーでかつ、アウトガスが極めて少ない粘着テープ（両面粘着タイプ［ＴＬ−６１７７−１２−ＧＡ］、基材レスタイプ［ＴＬ−６０７−ＧＡ、又はＴＬ−６０９−ＧＡ］、又は片面粘着タイプ［Ｈ−４１１７７−５０、Ｈ−４１０４１−５０、Ｈ−４１０５１−５０、又はＨ−４１０８１−５０］）を用いることもできる。 The display sticker of the present invention can be produced by applying a water-soluble adhesive for reducing or suppressing the generation of outgas on one side of a base material on which the generation of outgas is reduced or suppressed. Further, as the display sticker of the present invention, a commercially available product in which the generation of outgas is reduced or suppressed, for example, 3M (registered trademark) low outgas adhesive transfer tape (ATX203SF or ATX204SF) manufactured by 3M, or LINTEC. Silicone-free adhesive tape with extremely low outgas (double-sided adhesive type [TL-6177-12-GA], base material-less type [TL-607-GA, or TL-609-GA], or single-sided adhesive Types [H-41177-50, H-41041-50, H-41051-50, or H-41081-50]) can also be used.
本発明の表示用シールを貼付する箇所としては、哺乳類受精卵又は細胞の培養に用いる培養容器（マルチウエルプレート、培養皿［シャーレ、ディッシュ］等）の表面であればよく、哺乳類受精卵又は細胞と接触する培養容器の外側表面や、培養容器がマルチウエルプレートや培養皿の場合、培養容器の蓋上面の内側や蓋側周面の内側であってもよいが、通常は哺乳類受精卵又は細胞と接触しない培養容器の外側表面や、培養容器がマルチウエルプレートや培養皿の場合、培養容器の蓋上面の外側や蓋側周面の外側である。培養容器表面に貼付する表示用シールの枚数は、通常１枚であるが、本発明の表示用シールは、受精卵の胚発生や哺乳類細胞の増殖・生存に悪影響を及ぼさないという優れた効果を有するため、必要に応じて複数枚（全シールの総面積がシャーレの面積以内であればよく、例えば２〜１５枚の範囲内）であってもよい。 The display sticker of the present invention may be attached to the surface of a culture container (multi-well plate, culture dish [Petri dish, dish], etc.) used for culturing fertilized mammalian eggs or cells. The outer surface of the culture vessel that comes into contact with the culture vessel, or if the culture vessel is a multi-well plate or a culture dish, it may be inside the upper surface of the lid of the culture vessel or inside the peripheral surface on the lid side, but usually fertilized mammalian eggs or cells. The outer surface of the culture vessel that does not come into contact with the culture vessel, or when the culture vessel is a multi-well plate or a culture dish, it is the outside of the upper surface of the lid of the culture vessel or the outside of the peripheral surface on the lid side. The number of display stickers attached to the surface of the culture vessel is usually one, but the display stickers of the present invention have an excellent effect that they do not adversely affect the embryonic development of fertilized eggs or the proliferation and survival of mammalian cells. Therefore, if necessary, there may be a plurality of sheets (the total area of all the seals may be within the area of the petri dish, for example, within the range of 2 to 15 sheets).
以下に実施例により本発明を詳細に説明するが、本発明はこれらの実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.
１．胚発生における、表示用シールから放出されるアウトガスの影響の確認
胚発生における、表示用シールから放出されるアウトガスの影響を確認するために、以下の〔１〕〜〔３〕の手順の方法にしたがって検討を行った。
１−１ 方法
〔１〕アウトガスレスの水溶性粘着シールとして、アウトガスが極めて少ない粘着テープ（厚さ：０．０７５ｍｍ、製品番号：Ｈ−４１１７７−５０、LINTEC社製）を、縦８ｍｍ、横２８ｍｍとなるように切断したものを用いた。また、アウトガスのシールとして、ASONE社製のシール（縦：８ｍｍ、横：２８ｍｍ、厚さ：０．０７ｍｍ、製品番号：７２１５０）を用いた。これら２種類のシールを、培養ディッシュ（３００２、falcon社製）の蓋上面の内側に７枚、及び側周面内側に６枚貼り、培養ディッシュ（１００８、falcon社製）上に滴（ドロップ）状の培養液（４ｍｇ／ｍＬＢＳＡ［Bovine serum albumin］を含有するＭ１６培養液）（２０μＬ／滴）をのせ、その上をパラフィンオイル（OVOIL、VitroLife社製）（３ｍＬ／ディッシュ）で覆った後、蓋を除いた培養ディッシュ（３００２、falcon社製）の上に置いた（図１参照）。なお、コントロールとしてラベルシールを貼らない培養ディッシュを用いた実験を行った。
〔２〕滴状の培養液中にマウス受精卵を入れた後、インキュベーター（３７℃、５％Ｏ２、５％ＣＯ２、９０％Ｎ２）内で培養した。なお、マウス受精卵は、８週齢の雄ＢＤＦ１と雌ＢＤＦ１を用いて定法にしたがって体外受精を行い作製した。
〔３〕培養後２日目に、分割（卵割）した受精卵の数を算出し（表１、図２〜４の２日目参照）、培養後５日目に、胚盤胞に到達した受精卵の数を算出するとともに（表１、図２〜４の５日目参照）、Gardner分類により胚盤胞の発生ステージ（グレード１；初期胚盤胞［内腔が全体の１／２以下］、グレード２；胚盤胞［内腔が全体の１／２以上］、グレード３；完全胚盤胞、グレード４；拡張胚盤胞、グレード５；孵化中胚盤胞、及びグレード６；孵化後胚盤胞）を評価した（表２、図２〜４の５日目参照）。1. 1. Confirmation of the effect of outgas released from the display sticker on embryogenesis In order to confirm the effect of outgas released from the display seal on embryogenesis, the following procedure [1] to [3] Therefore, it was examined.
1-1 Method [1] As an outgas-less water-soluble adhesive seal, use an adhesive tape (thickness: 0.075 mm, product number: H-41177-50, manufactured by LINTEC) with extremely little outgas, 8 mm in length and 28 mm in width. The one cut so as to be used was used. Further, as an outgas seal, a seal manufactured by AS ONE (length: 8 mm, width: 28 mm, thickness: 0.07 mm, product number: 72150) was used. Seven of these two types of seals are attached to the inside of the upper surface of the lid of the culture dish (3002, manufactured by falcon) and six on the inside of the side peripheral surface, and dropped on the culture dish (1008, manufactured by falcon). Place the culture solution (M16 culture solution containing 4 mg / mL BSA [Bovine serum albumin]) (20 μL / drop), cover it with paraffin oil (OVOIL, manufactured by VitroLife) (3 mL / dish), and then cover it. It was placed on a culture dish (3002, manufactured by falcon) with the lid removed (see FIG. 1). An experiment was conducted using a culture dish without a label sticker as a control.
[2] Fertilized mouse eggs were placed in a drop-shaped culture medium and then cultured in an incubator (37 ° C., 5% O 2 , 5% CO 2 , 90% N 2 ). A mouse fertilized egg was prepared by in vitro fertilization using an 8-week-old male BDF1 and a female BDF1 according to a conventional method.
[3] The number of divided (split) fertilized eggs was calculated on the second day after culturing (see Table 1, the second day in FIGS. 2 to 4), and reached the blastocyst on the fifth day after culturing. Calculate the number of fertilized eggs (see Day 5 of Tables 1 and 2-4), and according to the Gardner classification, the developmental stage of blastocysts (grade 1; early blastocysts [the lumen is 1/2 of the total). Below], Grade 2; Blastocyst [more than 1/2 of the total lumen], Grade 3; Complete blastocyst, Grade 4; Expanded blastocyst, Grade 5; Hatching blastocyst, and Grade 6; Post-hatched blastocysts) were evaluated (see Table 2, days 5 of Figures 2-4).
１−２ 結果
まず、アウトガスの水溶性粘着シール（図５の「サンプル３」参照）をアウトガスレス処理したもの、すなわち、アウトガスレスの水溶性粘着シール（図５の「サンプル２」参照）におけるアウトガス量が検出感度以下であることを、ＧＣ−ＭＳを用いた解析により確認した。
アウトガスのシールを貼った培養ディッシュを用いて受精卵を培養した場合、シールを貼らなかった培養ディッシュを用いた場合と比べ、分割する受精卵の割合は９％と少なく、また、胚盤胞に到達した受精卵は全くなかった（表１参照）。一方、アウトガスレスの水溶性粘着シールを貼った培養ディッシュを用いて受精卵を培養した場合、シールを貼らなかった培養ディッシュを用いた場合と同様に、受精卵のすべてが分割し、胚盤胞に到達した受精卵の割合は約９０％と高く、そのほとんどが良好胚の目安とされているグレード３（完全胚盤胞）以上の胚盤胞であることが確認された（表１参照）。
以上の結果は、揮発性物質の発生が低減又は抑制された水溶性粘着シールは、受精卵の胚発生に悪影響を及ぼすことがないため、ヒト受精卵の培養に用いる培養容器表面に貼付するための表示用シールとして用いることができることを示している。1-2 Results First, the outgas-less water-soluble adhesive seal (see “Sample 3” in FIG. 5) was outgasless-treated, that is, the outgas in the outgasless water-soluble adhesive seal (see “Sample 2” in FIG. 5). It was confirmed by analysis using GC-MS that the amount was less than the detection sensitivity.
When fertilized eggs were cultivated using a culture dish with an outgas seal, the proportion of fertilized eggs to be divided was as low as 9% compared to the case with a culture dish without a seal, and the blastocysts No fertilized eggs arrived (see Table 1). On the other hand, when fertilized eggs are cultured using a culture dish with an outgasless water-soluble adhesive seal, all of the fertilized eggs are divided and blastocysts are divided, as in the case of using a culture dish without a seal. The proportion of fertilized eggs that reached the above was as high as about 90%, and it was confirmed that most of them were grade 3 (complete blastocysts) or higher blastocysts, which are considered to be good embryos (see Table 1). ..
The above results show that the water-soluble adhesive seal with reduced or suppressed generation of volatile substances does not adversely affect the embryonic development of fertilized eggs, and therefore is attached to the surface of the culture vessel used for culturing human fertilized eggs. It is shown that it can be used as a display sticker.
２．胚発生に悪影響を及ぼすアウトガスの同定
アウトガスのシールを培養ディッシュに貼付すると、シールから放出された揮発性物質が培養液中に溶解し、受精卵の胚発生に悪影響を及ぼすと考えられる。そこで、胚発生に悪影響を及ぼす揮発性物質を特定するために、以下の〔１〕〜〔３〕の手順の方法にしたがって検討を行った。
２−１ 方法
〔１〕実施例１で用いた２種類のシール（アウトガスレスの水溶性粘着シール及びアウトガスのシール）を、培養ディッシュ（３００２、falcon社製）の蓋上面の内側に７枚、及び側周面内側に６枚貼り、培養ディッシュ（１００８、falcon社製）上に滴状の培養液（４ｍｇ／ｍＬＢＳＡ［Bovine serum albumin］を含有するＭ１６培養液）（２０μＬ／滴）をのせ、その上をパラフィンオイル（OVOIL、VitroLife社製）（３ｍＬ／ディッシュ）で覆った後、蓋を除いた培養ディッシュ（３００２、falcon社製）の上に置いた（図１参照）。なお、コントロールとしてラベルシールを貼らない培養ディッシュを用いた実験を行った。
〔２〕インキュベーター（３７℃、５％Ｏ２、５％ＣＯ２、９０％Ｎ２）内で５日間インキュベートした後、培養液サンプルをヘッドスペースバイアルに回収した。
〔３〕培養液サンプルを６０℃で１時間加熱処理した後、測定装置（GCMS-TQ8030、島津社製）及びカラム（InertCap 1、ＧＬサイエンス社製）を用いたＧＣ−ＭＳにより解析した。なお、コントロールとしてアウトガスのシール（ASONE社製）自体を加熱処理したもの（表３の「シールサンプル」参照）も、ＧＣ−ＭＳにより解析した。2. Identification of outgas that adversely affects embryonic development When the outgas seal is attached to the culture dish, the volatile substances released from the seal dissolve in the culture medium, which is thought to have an adverse effect on embryonic development of fertilized eggs. Therefore, in order to identify volatile substances that adversely affect embryogenesis, studies were conducted according to the methods of the following procedures [1] to [3].
2-1 Method [1] Two types of seals (outgasless water-soluble adhesive seal and outgas seal) used in Example 1 were placed on the inside of the upper surface of the lid of the culture dish (3002, manufactured by falcon). And 6 sheets were pasted on the inside of the side peripheral surface, and a drop-shaped culture solution (M16 culture solution containing 4 mg / mL BSA [Bovine serum albumin]) (20 μL / drop) was placed on a culture dish (1008, manufactured by falcon). After covering it with paraffin oil (OVOIL, manufactured by VitroLife) (3 mL / dish), it was placed on a culture dish (3002, manufactured by falcon) without a lid (see FIG. 1). An experiment was conducted using a culture dish without a label sticker as a control.
[2] After incubating in an incubator (37 ° C., 5% O 2 , 5% CO 2 , 90% N 2 ) for 5 days, the culture solution sample was collected in 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 Co., Ltd.). As a control, a heat-treated outgas seal (manufactured by AS ONE) itself (see “Seal sample” in Table 3) was also analyzed by GC-MS.
２−２ 結果
アウトガスレスのシールを用いてインキュベートした場合、エタノール以外の揮発性物質は検出されなかったのに対して（表３の「培養液サンプル アウトガスレス」参照）、アウトガスのシールを用いてインキュベートした場合、エタノールに加えて２−プロパノールが検出された（表３の「培養液サンプル アウトガス」参照）。この結果から、胚発生に悪影響を及ぼす揮発性物質の１つとして、２−プロパノールが同定された。
なお、エタノールは、シールを貼付しなかった培養液サンプルでも検出されたことから（表３の「培養液サンプル −」参照）、アウトガスレスのシールで検出されたエタノールは、シールから放出されたものではなく、殺菌・消毒に用いた空気中のエタノールが混入したものと考えられる。2-2 Results When incubated with the outgasless seal, no volatile substances other than ethanol were detected (see “Culture sample outgasless” in Table 3), whereas the outgas seal was used. When incubated, 2-propanol was detected in addition to ethanol (see “Culture Sample Outgas” in Table 3). From this result, 2-propanol was identified as one of the volatile substances having an adverse effect on embryogenesis.
Since ethanol was also detected in the culture solution sample without the seal attached (see “Culture solution sample-” in Table 3), the ethanol detected in the outgasless seal was released from the seal. It is probable that ethanol in the air used for sterilization and disinfection was mixed in.
３．シールから放出される２−プロパノールの定量
アウトガスのシールを培養ディッシュに貼付すると、培養液中の２−プロパノールは検出感度以下であった。そこで、アウトガスのシール自体が、どの程度２−プロパノールを放出しているかについて、定量解析した。
３−１ 方法
実施例１で用いた２種類のシール（アウトガスレスの水溶性粘着シール及びアウトガスのシール）を、６０℃で１時間加熱処理した後、測定装置（GCMS-TQ8030、島津社製）及びカラム（InertCap 1、ＧＬサイエンス社製）を用いたＧＣ−ＭＳにより定量解析した。3. 3. Quantification of 2-propanol released from the seal When the outgas seal was attached to the culture dish, 2-propanol in the culture solution was below the detection sensitivity. Therefore, a quantitative analysis was performed on how much 2-propanol was released from the outgas seal itself.
3-1 Method Two types of seals (outgasless water-soluble adhesive seal and outgas seal) used in Example 1 were heat-treated at 60 ° C. for 1 hour, and then a measuring device (GCMS-TQ8030, manufactured by Shimadzu Corporation). Quantitative analysis was performed by GC-MS using a column (InertCap 1, manufactured by GL Science Co., Ltd.).
アウトガスのシールから放出される２−プロパノールは、０．０９３μＬ／ｇで検出されたのに対して、アウトガスレスのシールから放出される２−プロパノールは、検出感度以下（０．０８μＬ／ｇ未満）であった（表４参照）。 2-Propanol released from the outgas seal was detected at 0.093 μL / g, whereas 2-propanol released from the outgasless seal was below the detection sensitivity (less than 0.08 μL / g). (See Table 4).
本発明は、不妊治療に資するものである。 The present invention contributes to infertility treatment.
Claims (4)
Wherein the mammal is a human, the kit of claim 3.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015197772 | 2015-10-05 | ||
| JP2015197772 | 2015-10-05 | ||
| PCT/JP2016/004427 WO2017061097A1 (en) | 2015-10-05 | 2016-09-30 | Culturing-container display sticker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPWO2017061097A1 JPWO2017061097A1 (en) | 2018-07-12 |
| JP6774420B2 true JP6774420B2 (en) | 2020-10-21 |
Family
ID=58487359
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2017544183A Active JP6774420B2 (en) | 2015-10-05 | 2016-09-30 | Labeling sticker for culture container |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP6774420B2 (en) |
| TW (1) | TWI713598B (en) |
| WO (1) | WO2017061097A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6653908B1 (en) * | 2019-06-19 | 2020-02-26 | 有限会社品川通信計装サービス | Petri dish marking method for marking petri dishes |
| CN117402818B (en) * | 2023-12-15 | 2024-02-23 | 成都艾名迈德医学检验实验室有限公司 | Embryoid body packaging material, packaging device and preparation method of packaging device |
Family Cites Families (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4079659B2 (en) * | 2002-03-11 | 2008-04-23 | スリーエム イノベイティブ プロパティズ カンパニー | Low outgas pressure sensitive adhesive composition and pressure sensitive adhesive sheet |
| JP2005187674A (en) * | 2003-12-26 | 2005-07-14 | Toyo Ink Mfg Co Ltd | Adhesive composition and adhesive sheet using the same |
| TW200708305A (en) * | 2005-03-25 | 2007-03-01 | Nipro Corp | Freeze storage container and process for producing the same |
| US20110206927A1 (en) * | 2008-07-31 | 2011-08-25 | Lintec Corporation | Adhesive sheet |
| JP5364491B2 (en) * | 2009-07-24 | 2013-12-11 | リンテック株式会社 | Adhesive sheet |
| JP2011202126A (en) * | 2010-03-26 | 2011-10-13 | Nippon Synthetic Chem Ind Co Ltd:The | Adhesive sheet |
| JP2012060903A (en) * | 2010-09-14 | 2012-03-29 | Kajixx Kk | Adhesive sheet for multiwell plate and cell culture method |
| JP6291764B2 (en) * | 2013-09-25 | 2018-03-14 | 大日本印刷株式会社 | Label for cell culture container |
| JP2016099547A (en) * | 2014-11-25 | 2016-05-30 | 大日本印刷株式会社 | Display member, culture vessel and culture vessel kit |
-
2016
- 2016-09-30 TW TW105131647A patent/TWI713598B/en active
- 2016-09-30 JP JP2017544183A patent/JP6774420B2/en active Active
- 2016-09-30 WO PCT/JP2016/004427 patent/WO2017061097A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| JPWO2017061097A1 (en) | 2018-07-12 |
| TW201716567A (en) | 2017-05-16 |
| WO2017061097A1 (en) | 2017-04-13 |
| TWI713598B (en) | 2020-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Baert et al. | Mouse in vitro spermatogenesis on alginate-based 3D bioprinted scaffolds | |
| Fair et al. | Bovine oocyte diameter in relation to maturational competence and transcriptional activity | |
| You et al. | L-carnitine treatment during oocyte maturation improves in vitro development of cloned pig embryos by influencing intracellular glutathione synthesis and embryonic gene expression | |
| Hsu et al. | Development in vitro of mouse embryos from the two-cell egg stage to the early somite stage | |
| CN105154386B (en) | Human liver cell maintains for a long time and the special culture media and cultural method of propagation secondary culture | |
| CN104560870B (en) | A kind of method for preparing decidua mescenchymal stem cell | |
| Bassuner et al. | Auxin and root initiation in somatic embryos of Arabidopsis | |
| JP6536639B2 (en) | Stem cell culture medium | |
| KR20060032953A (en) | Cryopreservation of human blastocyst-derived stem cells by use of a closed straw vitrification method | |
| JP6774420B2 (en) | Labeling sticker for culture container | |
| US11913020B2 (en) | Method for improving stem cell migration using ethionamide | |
| JP7392935B2 (en) | Endometrium-like tissue and its production method | |
| Bashiri et al. | In vitro spermatogenesis in artificial testis: current knowledge and clinical implications for male infertility | |
| Yamamoto et al. | Probing teleost eye development by lens transplantation | |
| US20210386790A1 (en) | Use of mesenchymal stem cell sheets for preventing uterine scar formation | |
| Zhang et al. | Urothelial cell culture | |
| Tae et al. | Sterile filtered paraffin oil supports in vitro developmental competence in bovine embryos comparable to co-culture | |
| Seshagiri et al. | Enabling hamster embryo culture system: development of preimplantation embryos | |
| JP2004173635A (en) | Method for culturing oocyte and method for growing oocyte | |
| CN113930388A (en) | Chemical embryotoxicity prediction model and establishment method thereof | |
| TW201010709A (en) | Artificial kidney precursor and process for producing the same | |
| CN103224908A (en) | Tissue engineering material construction method based on amniotic mesenchymal stem cells | |
| Sakai et al. | Establishment of a mouse submandibular salivary gland organ culture | |
| CN101886059A (en) | Culture solution used for embryo vitro production and method for bovine embryo vitro production | |
| Reznikoff et al. | Altered growth patterns in vitro of human papillary transitional carcinoma cells. |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20180326 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20181211 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190116 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20190416 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190620 |
|
| C60 | Trial request (containing other claim documents, opposition documents) |
Free format text: JAPANESE INTERMEDIATE CODE: C60 Effective date: 20190620 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20190628 |
|
| C21 | Notice of transfer of a case for reconsideration by examiners before appeal proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C21 Effective date: 20190701 |
|
| A912 | Re-examination (zenchi) completed and case transferred to appeal board |
Free format text: JAPANESE INTERMEDIATE CODE: A912 Effective date: 20190830 |
|
| C211 | Notice of termination of reconsideration by examiners before appeal proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C211 Effective date: 20190903 |
|
| C22 | Notice of designation (change) of administrative judge |
Free format text: JAPANESE INTERMEDIATE CODE: C22 Effective date: 20200427 |
|
| C13 | Notice of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: C13 Effective date: 20200713 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200803 |
|
| C23 | Notice of termination of proceedings |
Free format text: JAPANESE INTERMEDIATE CODE: C23 Effective date: 20200824 |
|
| C03 | Trial/appeal decision taken |
Free format text: JAPANESE INTERMEDIATE CODE: C03 Effective date: 20200928 |
|
| C30A | Notification sent |
Free format text: JAPANESE INTERMEDIATE CODE: C3012 Effective date: 20200928 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20201002 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6774420 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |