JPH0538284A - Live cell preservative - Google Patents
Live cell preservativeInfo
- Publication number
- JPH0538284A JPH0538284A JP3173920A JP17392091A JPH0538284A JP H0538284 A JPH0538284 A JP H0538284A JP 3173920 A JP3173920 A JP 3173920A JP 17392091 A JP17392091 A JP 17392091A JP H0538284 A JPH0538284 A JP H0538284A
- Authority
- JP
- Japan
- Prior art keywords
- kestose
- cells
- cell preservative
- present
- live cell
- 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.)
- Granted
Links
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- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、生細胞を凍結状態また
は低温で長期間保存する際に必要な生細胞保存剤、特に
哺乳類、魚類の精子及び動物培養細胞の生細胞保存剤に
関するものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a viable cell preservative required for long-term preservation of viable cells in a frozen state or at a low temperature, particularly a preservative for viable cells of sperm of mammalian and fish and cultured cells of animal. is there.
【０００２】[0002]
【従来の技術】生細胞は、生活機能や生物学的活性を有
しており多方面で利用価値があるが、温度の高い条件下
では代謝あるいは変性のために経時的に変化、生活機能
や生物学的活性が低下ないし消失してしまう。このよう
な生細胞の長期保存には凍結保存が適すると考えられ、
例えば細胞内凍害保護物質と細胞外凍害保護物質の共働
効果を図った特公昭６０−２９４７１号や、凍結保護剤
としてメチルセルロースを使用した特開昭６３−２１６
４７６号や、凍結保護剤としてベタインを使用した特開
平２−４２２号等、今日まで多くの研究が行われてい
る。BACKGROUND OF THE INVENTION Living cells have a living function and biological activity and are useful in many fields, but under high temperature conditions they change over time due to metabolism or denaturation, Biological activity decreases or disappears. Cryopreservation is considered to be suitable for such long-term preservation of live cells.
For example, Japanese Examined Patent Publication No. 60-29471, which has a synergistic effect of an intracellular frost damage-protecting substance and an extracellular frost damage-protecting substance, and JP-A-63-216 using methylcellulose as a cryoprotectant.
Many studies have been conducted to date, such as Japanese Patent Laid-Open No. 422/422, which uses betaine as a cryoprotectant.
【０００３】一般に、生細胞に対する凍害防止効果を示
す物質の条件として次の項目が挙げられる。 中性物質（ｎｅｕｔｒａｌ ｓｏｌｕｔｅ）であるこ
と。 低分子物質（即ち、細胞に対する透過性が高い物質）
であること。 束一性（ｃｏｌｌｉｇａｔｉｖｅ ｐｒｏｐｅｒｔ
ｙ，即ち、水素結合を作り易く、共晶点が低い性質）を
持っていること。In general, the following items can be mentioned as conditions for substances exhibiting an antifreezing effect on living cells. It must be a neutral substance. Low molecular weight substances (that is, substances with high permeability to cells)
To be. Colligative property
y, that is, the property of easily forming hydrogen bonds and having a low eutectic point).
【０００４】高濃度でも細胞に対する毒性が低いこ
と。 現在のところ、これらの条件を満たし広く使用されてい
る物質に、グリセリンやＤＭＳＯ（ｄｉｍｅｔｈｙｌｓ
ｕｌｆｏｘｉｄｅ）がある。しかし、グリセリンについ
ては単独で使用することは稀で、動物、例えば牛の精子
凍結保存希釈液にグリセリンと糖との混合液が広く用い
られている。Low toxicity to cells even at high concentration. At present, glycerin and DMSO (dimethyls) are widely used substances that satisfy these conditions.
uloxide). However, glycerin is rarely used alone, and a mixed solution of glycerin and sugar is widely used as a frozen preservation solution of spermatozoa of animals such as cattle.
【０００５】そのメリットは、室温で一度の希釈が可能
であること、グリセリン平衡時間が短縮されること、お
よび広い範囲の凍結速度が適用できる等があげられる。
また糖類の凍害防止効果は、５、６炭糖（Ｐｅｎｔｏｓ
ｅ，Ｈｅｘｏｓｅ）よりも２，３糖類の方が優れている
ことから、従来は主に３糖類であるラフィノースが用い
られてきた。The merit is that it can be diluted once at room temperature, the glycerin equilibration time can be shortened, and a wide range of freezing rate can be applied.
In addition, the anti-freezing effect of sugar is 5 or 6 carbon sugar (Pentos
Since the 2,3 sugars are superior to e, Hexose), raffinose, which is a trisaccharide, has been mainly used conventionally.
【０００６】[0006]
【発明が解決しようとする課題】しかしながら、ラフィ
ノースの化学合成は不可能で、天然に存在する植物例え
ばビートなどから抽出、精製しなければ入手できない。
その供給量は限られているので、高価であった。またＤ
ＭＳＯは、有機溶剤であるため水洗い等の操作が必要と
なることがあり、一般的に取扱が繁雑でこれにかわるも
のが強く望まれていた。However, the chemical synthesis of raffinose is impossible and cannot be obtained unless it is extracted and purified from naturally occurring plants such as beet.
It was expensive because its supply was limited. Also D
Since MSO is an organic solvent, it may require operations such as washing with water. In general, handling is complicated and an alternative to this has been strongly desired.
【０００７】本出願人は、ショ糖を原料として高純度の
１−ケストースを安価に製造する方法を既に出願してお
り（特願平２−２２４３１２号）、また、この１−ケス
トースに生細胞の保存効果があることの知見を得た。そ
こで本発明は、１−ケストースを用いた生細胞保存剤を
提供することを目的とする。The present applicant has already applied for a method for inexpensively producing high-purity 1-kestose using sucrose as a raw material (Japanese Patent Application No. 2-224312), and the 1-kestose is a living cell. It was found that there is a preservative effect. Therefore, an object of the present invention is to provide a live cell preservative containing 1-kestose.
【０００８】[0008]
【課題を解決するための手段】前記問題点を解決するた
めに本発明は、１−ケストースを有効成分として含有す
ることを特徴とする生細胞保存剤とするものである。ま
た本発明は、１−ケストース濃度が０．１〜２０％（Ｗ
／Ｖ）に調整されたことを特徴とする生細胞保存剤とす
るものである。To solve the above problems, the present invention provides a viable cell preservative characterized by containing 1-kestose as an active ingredient. In the present invention, the 1-kestose concentration is 0.1 to 20% (W
/ V), which is a viable cell preservative.
【０００９】また本発明は、哺乳動物の精子、魚類の精
子または動物培養細胞から選ばれた生細胞の保存に１−
ケストースを用いることを特徴とする生細胞保存剤とす
るものである。１−ケストースはいわゆるフラクトオリ
ゴ糖の一種であり、その構造はグルコース（Ｇ）とフラ
クトース（Ｆ）が結合したシュークロース（ＧＦ）のフ
ラクトース部分に、β−２，１結合によりフラクトース
が１分子結合したものであって、Ｏ−α−Ｄ−グルコピ
ラノシル−（１→２）−Ｏ−β−Ｄ−フラクトフラノシ
ル−（１→２）−Ｏ−β−Ｄ−フラクトフラノシドであ
る。その分子式は、Ｃ18Ｈ32Ｏ16であり、その分子量は
５０４である。次に１−ケストースの構造式を示す。The present invention also relates to the preservation of living cells selected from mammalian sperm, fish sperm or animal cultured cells.
The present invention provides a live cell preservative characterized by using kestose. 1-Kestose is a kind of so-called fructo-oligosaccharides, and its structure is that one molecule of fructose is bound to the fructose portion of sucrose (GF), which is bound to glucose (G) and fructose (F), by β-2,1 bond. Which is O-α-D-glucopyranosyl- (1 → 2) -O-β-D-fructofuranosyl- (1 → 2) -O-β-D-fructofuranoside. Its molecular formula is C 18 H 32 O 16 and its molecular weight is 504. Next, the structural formula of 1-kestose is shown.
【００１０】[0010]
【化１】 [Chemical 1]
【００１１】従来のフラクトオリゴ糖の用途は特公昭５
９−５３８３４号に開示されているビフィズス活性や特
開昭５９−１１０６２１号に開示されている利尿剤等が
あるがいずれもフラクトオリゴ糖の混合物、即ち１ケス
トース（ＧＦ2 ）、ニストース（ＧＦ3 ）、フラクトフ
ラノシルニストース（ＧＦ4 ）の混合物であった。１−
ケストースの製造法は特開平２−１６３０９３号や特開
平２−２４９４９３号に開示されており、ショ糖にスコ
プラリオプシス・ブレビカウリス（Ｓｃｏｐｕｌａｒｉ
ｏｐｓｉｓ ｂｒｅｖｉｃａｕｌｉｓ）の生産するフラ
クトシルトランスフェラーゼを作用させることによって
得ることができる。また、ショ糖を原料として高純度の
１−ケストースを安価に製造する方法は、本出願人が特
願平２−２２４３１２号として既に出願している。Conventional fructooligosaccharides are used in Japanese Patent Publication No.
There are bifidos activity disclosed in JP-A-9-53834 and diuretics disclosed in JP-A-59-110621, but both are mixtures of fructooligosaccharides, that is, 1 kestose (GF 2 ) and nystose (GF 3 ). , A mixture of fructofuranosyl nystose (GF 4 ). 1-
The method for producing kestose is disclosed in JP-A Nos. 2-163093 and 2-2494943, and sucrose is added to Scopulariopsis brevicauris (Scopulari).
It can be obtained by acting a fructosyl transferase produced by Opsis brevicus. Further, a method for inexpensively producing 1-kestose of high purity using sucrose as a raw material has already been filed by the applicant as Japanese Patent Application No. 2-24312.
【００１２】この生産法によると工業的に１−ケストー
スが単品でしかも高純度（９９．９％）で大量に得られ
る。この高純度１−ケストースが生細胞保存剤として優
れた効果を有していることの知見を得、本発明を完成す
るに至った。本発明において使用する１−ケストース
は、凍結する生細胞に適した濃度で使用し、その濃度は
０．１〜２０％（Ｗ／Ｖ）の範囲内である。また本発明
の１−ケストースは、生細胞保存剤の組成の一部または
全部として使用することができる。例えば、哺乳動物の
精子の凍結保存においては、既存の生細胞保存剤（糖含
有グリセリン・卵白懸濁液）中の１組成物であるラフィ
ノースの代替として０．１〜０．５％（Ｗ／Ｖ）使用す
ることができる。According to this production method, 1-kestose is industrially obtained as a single product and in high purity (99.9%) in a large amount. The present inventors have found that this highly pure 1-kestose has an excellent effect as a preservative for live cells, and have completed the present invention. The 1-kestose used in the present invention is used at a concentration suitable for freezing living cells, and the concentration is within the range of 0.1 to 20% (W / V). The 1-kestose of the present invention can be used as a part or the whole of the composition of the preservative for live cells. For example, in the cryopreservation of mammalian sperm, 0.1-0.5% (W / W) is used as a substitute for raffinose, which is one composition in an existing preservative for living cells (sugar-containing glycerin / egg white suspension). V) Can be used.
【００１３】また、魚類の精子や動物培養細胞において
は１−ケストースを３〜２０％（Ｗ／Ｖ）に調整し、そ
のまま単独で凍結保存剤として使用することができる。
以下に、実施例をもって本発明を説明するが、これはあ
くまでも例示であって、本発明はこれに限定されるもの
ではない。In fish spermatozoa and animal cultured cells, 1-kestose can be adjusted to 3 to 20% (W / V) and used alone as a cryopreservative.
Hereinafter, the present invention will be described with reference to Examples, but these are merely examples and the present invention is not limited thereto.
【００１４】[0014]
【実施例１】牛の精子の凍結保存について、本発明の１
−ケストースを含む生細胞保存剤を使用し、その保存効
果の実験を行なった。対照として、従来から使用されて
いる生細胞保存剤であるラフィノースを含む組成物から
なる生細胞保存剤を使用した。生細胞保存剤に関して
は、対照と本発明のものは１−ケストースとラフィノー
スが代わっただけであり、その他の成分は全て同じであ
る。Example 1 Regarding cryopreservation of bovine spermatozoa
-Using a live cell preservative containing kestose, experiments of its preservative effect were performed. As a control, a live cell preservative composed of a composition containing raffinose, which is a conventionally used live cell preservative, was used. Regarding the viable cell preservative, the control and the one of the present invention only have 1-kestose and raffinose in place, and all other components are the same.
【００１５】実験に使用した凍結保存液の組成を次の表
１に示す。The composition of the cryopreservation liquid used in the experiment is shown in Table 1 below.
【００１６】[0016]
【表１】 [Table 1]
【００１７】牛の精子の凍結保存の実験条件は、凍結温
度を−１９６℃で、６か月間保存した後、解凍して精子
活力を調べた。精子の活力の判定は、顕微鏡にて精子を
観察し、勢い良く前進するもの（＋＋＋）、前進するも
の（＋＋）、頭を動かすもの（＋）とし、（＋＋＋）を
精子活力の数字（％）とした。その結果を次の表２に示
す。The experimental conditions for the cryopreservation of bovine spermatozoa were that the freezing temperature was -196 ° C., the cells were stored for 6 months, and then thawed to examine the sperm vitality. The sperm vitality is determined by observing the sperm under a microscope, and those that vigorously move forward (+++), those that move forward (++), and those that move the head (+), and (+++) is the number (%) of sperm vitality. ). The results are shown in Table 2 below.
【００１８】[0018]
【表２】 [Table 2]
【００１９】表２によれば、本発明の１−ケストースを
含む生細胞保存剤は、従来のラフィノースを含む生細胞
保存剤と同等の効果を示すことがわかる。From Table 2, it is understood that the living cell preservative containing 1-kestose of the present invention exhibits the same effect as that of the conventional living cell preservative containing raffinose.
【００２０】[0020]
【実施例２】サクラマス（Ｏｎｃｏｒｈｙｎｃｈｕｓ
ｍａｓｏｕ）の精子２０μｌに１−ケストースまたはラ
フィノースの水溶液、或いは、ＤＭＳＯ溶液（２５ｍＭ
Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５に溶解したもの）を８
０μｌ加え、最終濃度を１−ケストース、ラフィノース
は６０μＭ、ＤＭＳＯ溶液は２％となるようにして各保
存溶液を調製した。なお、１−ケストースは純度９９．
９％のものを用いた。このように調製した各保存溶液５
０μｌを、ドライアイス上にドリルで作った直径５ｍｍ
の窪みに乗せて急速凍結して、凍結した精子ペレットを
作成した。Example 2 Cherry salmon (Oncorhynchus)
20 μl of spermatozoa (masou), an aqueous solution of 1-kestose or raffinose, or a DMSO solution (25 mM
Tris-HCl, dissolved in pH 7.5) 8
Each stock solution was prepared by adding 0 μl and adjusting the final concentrations to 1-kestose, raffinose of 60 μM, and DMSO solution of 2%. The purity of 1-kestose is 99.
9% was used. 5 stock solutions prepared in this way
Drilled 0 μl on dry ice, diameter 5 mm
The frozen sperm pellet was prepared by placing it in the hollow of the and rapidly freezing.
【００２１】この精子ペレットを−７０℃で保存し、２
日後と５１日後に４℃で解凍し、顕微鏡下で運動性を調
べた。採精直後の運動性（運動開始から停止までの時
間）を１００とした。その結果を次の表３に示す。The sperm pellet was stored at -70 ° C.
Thaw at 4 ° C. after day and 51 days and examined for motility under a microscope. The motility immediately after the insemination (time from the start of exercise to the stop) was set to 100. The results are shown in Table 3 below.
【００２２】[0022]
【表３】 [Table 3]
【００２３】表３からは、本発明の１−ケストースを単
独使用した生細胞保存剤が優れた保存効果を有すること
がわかる。It can be seen from Table 3 that the live cell preservative of the present invention, which uses 1-kestose alone, has an excellent preservation effect.
【００２４】[0024]
【実施例３】培養細胞としてガン細胞由来のＨｅｌａ細
胞を用い、５％牛胎児血清を添加したＬ−１５培地にて
３７℃で単層を形成するまで培養した後、単層を形成し
たＨｅｌａ細胞をトリプシンで消化することによりフラ
スコの底に付いたＨｅｌａ細胞を剥がす。これを滅菌し
たピペットで分取し、３×１０5 ／ｍｌになるように動
物細胞培養用の一般的培地であるＬ−１５培地に懸濁し
た。これに１／５量の１−ケストース、グルコース、シ
ュークロース、ＤＭＳＯを最終濃度が１０％になるよう
に加えた。なお、１−ケストースは純度９９．９％のも
のを用いた。これを４℃、−２０℃、−８０℃で１６日
間保存後、室温解凍し生細胞数をトリパンブルー染色し
たのち血球計算板で生細胞の数を測定した。なお、該染
色法を使用すると生細胞の核を染めるが、死細胞の核は
染めないので、凍結保存後の生存率がわかる。その結果
を次の表４に示す。[Example 3] Hela cells derived from cancer cells were used as cultured cells, and the cells were cultured in L-15 medium supplemented with 5% fetal bovine serum at 37 ° C until a monolayer was formed. The Hela cells attached to the bottom of the flask are detached by digesting the cells with trypsin. This was collected with a sterilized pipette and suspended in L-15 medium, which is a general medium for culturing animal cells, at 3 × 10 5 / ml. To this was added 1/5 amount of 1-kestose, glucose, sucrose, DMSO to a final concentration of 10%. The 1-kestose used had a purity of 99.9%. This was stored at 4 ° C, -20 ° C, and -80 ° C for 16 days, thawed at room temperature, stained with trypan blue for the number of viable cells, and then the number of viable cells was measured with a hemocytometer. When the staining method is used, the nuclei of living cells are stained, but the nuclei of dead cells are not stained, so that the survival rate after cryopreservation can be known. The results are shown in Table 4 below.
【００２５】[0025]
【表４】 [Table 4]
【００２６】表４からは、本発明の１−ケストースを単
独使用した生細胞保存剤は４℃、−２０℃、−８０℃の
何れにおいても優れた保存効果を示すことがわかる。It can be seen from Table 4 that the live cell preservative of the present invention, which uses 1-kestose alone, exhibits an excellent preservation effect at any of 4 ° C, -20 ° C and -80 ° C.
【００２７】[0027]
【実施例４】培養細胞としてアフリカミドリザルの腎細
胞のＶｅｒｏ細胞を用い、５％牛胎児血清を添加したＬ
−１５培地にて３７℃で培養後、単層を形成したＶｅｒ
ｏ細胞をトリプシンで消化し、１．５×１０5 ／ｍｌに
なるようにＬ−１５培地に懸濁した。これに１／５量の
１−ケストース、グルコース、シュークロース、ＤＭＳ
Ｏを最終濃度が１０％になるように加えた。なお、１−
ケストースは純度９９．９％のものを用いた。これを４
℃、−２０℃、−８０℃で１６日間保存後、室温解凍し
生細胞数をトリパンブルー染色したのち血球計算板で測
定した。その結果を次の表５に示す。Example 4 Vero cells of African green monkey kidney cells were used as culture cells, and L supplemented with 5% fetal bovine serum was used.
After culturing in -15 medium at 37 ° C, Ver formed a monolayer
The o cells were digested with trypsin and suspended in L-15 medium at 1.5 × 10 5 / ml. 1/5 amount of 1-kestose, glucose, sucrose, DMS
O was added to a final concentration of 10%. In addition, 1-
The kestose used had a purity of 99.9%. This 4
After storing at 16 ° C, -20 ° C, and -80 ° C for 16 days, the cells were thawed at room temperature, stained with trypan blue for viable cell count, and then measured with a hemocytometer. The results are shown in Table 5 below.
【００２８】[0028]
【表５】 [Table 5]
【００２９】表５からは、本発明の１−ケストースを単
独使用した生細胞保存剤は４℃、−８０℃で優れた保存
効果を示すことがわかる。以上、本実施例を実験データ
に基づいて説明したが、本発明は上記実施例に限定され
ず、本発明の趣旨に基づき、種々の変形が可能である。
例えば、１−ケストースの純度を９９．９％のものにつ
いて実験したが、もっと低純度のものでも使用可能であ
ることは言うまでもない。From Table 5, it can be seen that the living cell preservative of the present invention, which uses 1-kestose alone, exhibits an excellent preservation effect at 4 ° C and -80 ° C. Although the present embodiment has been described above based on the experimental data, the present invention is not limited to the above embodiment, and various modifications can be made based on the spirit of the present invention.
For example, 1-kestose having a purity of 99.9% was tested, but it goes without saying that a lower purity can be used.
【００３０】[0030]
【発明の効果】本発明の生細胞保存剤は、１−ケストー
スを単独で使用しても、または他の生細胞保存剤と併用
しても、何れもの場合でも優れた保存効果を有する。本
発明の生細胞保存剤は、凍結保存だけではなく、凍結し
ない程度の低温での保存にも効果を有する。INDUSTRIAL APPLICABILITY The live cell preservative of the present invention has an excellent preservative effect regardless of whether 1-kestose is used alone or in combination with other live cell preservatives. The live cell preservative of the present invention is effective not only for cryopreservation but also for preservation at a low temperature at which it does not freeze.
【００３１】１−ケストースは微生物工業により大量生
産ができるので、安価に生細胞保存剤を提供することが
できる。Since 1-kestose can be mass-produced by the microbial industry, a viable cell preservative can be provided at low cost.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 竹田 博幸 北海道札幌市中央区北４条西１丁目３番地 ホクレン農業協同組合連合会内 (72)発明者 古賀 裕 北海道札幌市中央区北４条西１丁目３番地 ホクレン農業協同組合連合会内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroyuki Takeda 1-3, Kita 4-jo Nishi, Chuo-ku, Sapporo-shi, Hokkaido Hokuren Agricultural Cooperative Federation (72) Inventor Yu Koga Kita 4-jo Nishi, Chuo-ku, Sapporo-shi, Hokkaido 1-3, Hokuren Agricultural Cooperative Federation
Claims (3)
ることを特徴とする生細胞保存剤。1. A viable cell preservative containing 1-kestose as an active ingredient.
（Ｗ／Ｖ）に調整されたことを特徴とする請求項１記載
の生細胞保存剤。2. The 1-kestose concentration is 0.1 to 20%.
The live cell preservative according to claim 1, which is adjusted to (W / V).
培養細胞から選ばれた生細胞の保存に用いることを特徴
とする請求項１または２記載の生細胞保存剤。3. The viable cell preservative according to claim 1 or 2, which is used for preserving live cells selected from mammalian sperm, fish sperm or animal cultured cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3173920A JPH07114692B2 (en) | 1991-07-15 | 1991-07-15 | Live cell preservative |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3173920A JPH07114692B2 (en) | 1991-07-15 | 1991-07-15 | Live cell preservative |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0538284A true JPH0538284A (en) | 1993-02-19 |
| JPH07114692B2 JPH07114692B2 (en) | 1995-12-13 |
Family
ID=15969530
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3173920A Expired - Fee Related JPH07114692B2 (en) | 1991-07-15 | 1991-07-15 | Live cell preservative |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07114692B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5575190A (en) * | 1993-10-04 | 1996-11-19 | Jatco Corporation | Control valve arrangement of automatic transmission |
| WO2003086072A1 (en) * | 2002-03-28 | 2003-10-23 | Meiji Seika Kaisha, Ltd. | Composition for storing organ and method of storing organ |
| WO2017159737A1 (en) * | 2016-03-16 | 2017-09-21 | 一般社団法人家畜改良事業団 | Diluent for sperm and method for preserving sperm using same |
-
1991
- 1991-07-15 JP JP3173920A patent/JPH07114692B2/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5575190A (en) * | 1993-10-04 | 1996-11-19 | Jatco Corporation | Control valve arrangement of automatic transmission |
| WO2003086072A1 (en) * | 2002-03-28 | 2003-10-23 | Meiji Seika Kaisha, Ltd. | Composition for storing organ and method of storing organ |
| JPWO2003086072A1 (en) * | 2002-03-28 | 2005-08-18 | 明治製菓株式会社 | Organ preservation composition and organ preservation method |
| AU2003236297B2 (en) * | 2002-03-28 | 2008-12-11 | Meiji Seika Kaisha, Ltd. | Composition for storing organ and method of storing organ |
| US7476660B2 (en) | 2002-03-28 | 2009-01-13 | Meiji Seika Kaisha, Ltd. | Composition and method for organ preservation |
| JP4570877B2 (en) * | 2002-03-28 | 2010-10-27 | 明治製菓株式会社 | Organ preservation composition and organ preservation method |
| WO2017159737A1 (en) * | 2016-03-16 | 2017-09-21 | 一般社団法人家畜改良事業団 | Diluent for sperm and method for preserving sperm using same |
| JPWO2017159737A1 (en) * | 2016-03-16 | 2019-01-24 | 一般社団法人家畜改良事業団 | Sperm dilution and method for preserving sperm using the same |
| US11185068B2 (en) | 2016-03-16 | 2021-11-30 | Livestock Improvement Association Of Japan, Inc. | Diluent for sperm and method for preserving sperm using same |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07114692B2 (en) | 1995-12-13 |
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