JP4348458B2 - Serum-free early embryo culture - Google Patents
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本発明は初期胚組織を培養するための培養液であって、特に血清を含まず体外受精胚やクローン胚の構築・発育をも可能な培地たり得る無血清初期胚培養液に関するものである。 The present invention relates to a culture medium for culturing early embryonic tissue, and particularly to a serum-free early embryo culture medium that does not contain serum and can be a medium capable of constructing and developing in vitro fertilized embryos and cloned embryos.
従来、牛の体外受精卵子を培養する際には、胚組織培養液に血清をエネルギー源として添加していた。また、体外受精卵子の胚発育効率を改善するためには、体外受精直後から初期胚の体外発生培養に、各種体細胞との共培養を実施することが普通で、この共培養も血清培地中で行われていた。例えば非特許文献１には、過***処理された牛から得られた５〜８細胞の胚が卵管細胞と共培養された結果が示されている。
しかしながら、血清の化学的組成は不明であるとともにそのロットによって胚に及ぼす生物活性は大きく変動するなどの難点がある。また、血清には脂肪が含まれるため、胚組織に脂肪が蓄積されてしまうことで例えば巨大児が発生してしまうというような不具合も生じている。しかも、血清は高価であるため多量に使用することが困難である場合も多く、血清がウイルスなどに汚染されていることも多い。このようなウイルスは胚の発育に悪影響を与え、作出効率にも影響が及ぶ場合もある。
そこで、無血清培地による牛胚組織の培養に関する研究が行われるようになり、例えば非特許文献２では、過***処理された牛から１〜２細胞の胚や卵を外科的な処置により取り出して、胚をグルコースフリーかつ無血清で共培養する実験について開示している。この非特許文献２においては、この胚が胚盤胞まで成長したことが紹介されている。
さらに、特許文献１においては「牛胚の体外発生方法」として、無血清培地中メタロプロテイナーゼインヒビターの存在下で牛受精卵を体外培養することを特徴とする胚発生方法が開示されている。この発明では、顆粒膜細胞を無血清培地中で長期にわたって培養することに成功し、その結果、培養上清中に含まれる有効成分として前述のメタロプロテイナーゼインヒビター（以下、ＴＩＭＰ[Tissue Inhibitor of Metallo Proteinase]と略す。）を見出して、その存在下で牛受精卵を培養するものである。具体的には、基礎培地に市販の組織培養培地ＴＣＭ１９９（日水製薬製）を用い、これにＴＩＭＰを添加して牛受精卵を体外培養している。
本発明によれば、組成が不明である血清を用いることなく、組成が明らかな無血清培地を用いて胚の発生を促すことが可能である。
Conventionally, when culturing bovine in vitro fertilized ova, serum has been added to the embryo tissue culture medium as an energy source. In order to improve the embryo development efficiency of in vitro fertilized eggs, it is common to perform co-culture with various somatic cells immediately after in vitro fertilization for in vitro development of early embryos. It was done in. For example, Non-Patent Document 1 shows the result of co-culturing 5-8 cell embryos obtained from superovulated cattle with oviduct cells.
However, the chemical composition of serum is unknown, and the biological activity on embryos varies greatly depending on the lot. In addition, since serum contains fat, there is a problem that, for example, a giant baby is generated due to the accumulation of fat in the embryonic tissue. Moreover, since serum is expensive, it is often difficult to use a large amount of serum, and the serum is often contaminated with viruses. Such viruses can adversely affect embryo development and can affect production efficiency.
Therefore, research on the culture of bovine embryo tissues using a serum-free medium has been conducted. For example, in Non-Patent Document 2, 1-2 cell embryos and eggs are taken out from a superovulated cow by surgical treatment. Discloses experiments in which embryos are co-cultured in a glucose-free and serum-free manner. In this non-patent document 2, it is introduced that this embryo has grown to a blastocyst.
Furthermore, Patent Document 1 discloses a method for developing an embryo characterized by in vitro culture of a fertilized bovine egg in the presence of a metalloproteinase inhibitor in a serum-free medium as a “method for in vitro generation of a cow embryo”. In the present invention, granulosa cells have been successfully cultured for a long time in a serum-free medium, and as a result, the metalloproteinase inhibitor (hereinafter referred to as TIMP [Tissue Inhibitor of Metallo Proteinase) is used as an active ingredient contained in the culture supernatant. ] Is abbreviated.), And cow fertilized eggs are cultured in the presence. Specifically, a commercially available tissue culture medium TCM199 (manufactured by Nissui Pharmaceutical) is used as a basal medium, and TIMP is added thereto to in vitro culture bovine fertilized eggs.
According to the present invention, it is possible to promote embryo development using a serum-free medium with a clear composition without using serum with an unknown composition.
しかしながら、上述の非特許文献１に開示された発明においては、前述のとおり血清が使用されており、その化学的組成が不明で品質にもばらつきがありウイルスによる汚染などの可能性もあるという課題があった。また、これらの原因によって正常な胚の発生阻害や胎仔の過大化などの可能性もあるという課題もあった。
非特許文献２に開示された発明においては、卵管細胞と共培養する必要があり、胚組織の培養に手間がかかるという課題があった。
さらに、特許文献１に開示された発明においては、ＴＩＭＰが発育促進の機能を発揮するものの、ＴＩＭＰが高価であり広く普及するにはコスト的な課題があった。
本発明はかかる従来の事情に対処してなされたものであり、組成が明確で血清を添加した培地に係る不都合な要因、例えばウイルスへの感染や正常な胚発生の阻害、胎仔の過大化などを排除可能な無血清の培養液であって、比較的簡単な組成で取扱いも容易でしかも安価に製造することが可能な無血清初期胚培養液を提供することを目的とする。
However, in the invention disclosed in Non-Patent Document 1 described above, serum is used as described above, the chemical composition of which is unknown, quality varies, and there is a possibility of contamination by viruses. was there. In addition, there is a problem that there is a possibility that the development of normal embryos and the fetus become excessive due to these causes.
In the invention disclosed in Non-Patent Document 2, there is a problem that it is necessary to co-culture with oviduct cells, and it takes time to culture embryo tissues.
Furthermore, in the invention disclosed in Patent Document 1, although TIMP exerts the function of promoting growth, TIMP is expensive and has a problem in terms of cost.
The present invention has been made in response to such a conventional situation, and has an unfavorable factor relating to a medium with a clear composition and supplemented with serum, such as inhibition of virus infection and normal embryogenesis, fetal overgrowth, etc. It is an object of the present invention to provide a serum-free culture medium that can eliminate the above-mentioned serum-free embryo culture medium that is relatively easy to handle and that can be manufactured at low cost.
請求項１に記載の発明である無血清初期胚培養液においては、含有成分が特許請求の範囲の表１で表される濃度であることを特徴とするものである。
上記構成の無血清初期胚培養液においては、塩化カルシウムに代えられるヘミカルシウムラクテートが、カルシウム濃度を維持し、かつエネルギー源として胚の発育を支持するように作用する。また、アラニン、グリシン、タウリン、インスリンというアミノ酸構成成分が添加されることによって、エネルギー源となると同時に解毒作用やｐＨの調整を促進させてタンパク合成を活発化させる作用を有する。
なお、濃度の範囲として±１０％と記載しているのは、特にその数値のみに限定するものではないという意味があるのと同時に、試験などを実施した際には培養液を調製する際に、略±１０％程度の液量測定誤差が生じており、そのような範囲にあっても試験結果では略同等の効果を得ていることが根拠となっている。
The serum-free early embryo culture solution according to the first aspect of the present invention is characterized in that the contained component has a concentration shown in Table 1 of the claims.
In the serum-free early embryo culture medium having the above-described configuration, hemi-calcium lactate substituted for calcium chloride acts to maintain the calcium concentration and support embryo development as an energy source. In addition, the addition of amino acid components such as alanine, glycine, taurine, and insulin serves as an energy source and at the same time promotes detoxification and pH adjustment to activate protein synthesis.
In addition, the description of ± 10% as the concentration range means that it is not particularly limited only to the numerical value, and at the same time, when a culture solution is prepared when a test or the like is performed. The liquid amount measurement error of about ± 10% occurs, and it is based on the fact that the test result obtains substantially the same effect even in such a range.
本発明の無血清初期胚培養液においては、成分の明確な無血清培養液であるため血清を用いた培地のようなウイルス汚染や品質のばらつきがなく、正常な胚発生や胚組織の培養を行うことができる。また、取扱いが容易で、比較的簡単な組成であることから製造コストも抑制することができる。 In the serum-free early embryo culture solution of the present invention, since it is a serum-free culture solution with a clear component, there is no virus contamination or quality variation like a medium using serum, and normal embryo development and embryo tissue culture are performed. It can be carried out. Moreover, since it is easy to handle and has a relatively simple composition, the manufacturing cost can be reduced.
以下に本発明の実施の形態に係る無血清初期胚培養液について表２を参照しながら説明する。
表２の左欄Ａは、培養液ＳＯＦ(Synthetic Oviduct Fluid)の組成表であり、右欄Ｂは本発明の実施の形態に係る無血清初期胚培養液の組成表である。それぞれの組成は１００ｍｌ当たりに含まれる成分量を示している。
The serum-free early embryo culture medium according to the embodiment of the present invention will be described below with reference to Table 2.
The left column A of Table 2 is a composition table of the culture fluid SOF (Synthetic Oviduct Fluid), and the right column B is a composition table of the serum-free early embryo culture solution according to the embodiment of the present invention. Each composition shows the amount of components contained per 100 ml.
表２の左欄及び右欄において、ＢＳＡは牛血清アルブミンを意味している。本実施の形態に係る無血清初期胚培養液では、培養液ＳＯＦに含まれていた塩化カルシウムに代えてヘミカルシウムラクテートを０．５４ｇ含んでいる。また、その他ＳＯＦに含まれずに本実施の形態に係る無血清初期胚培養液に含まれる成分として、ＰＶＡ（ポリビニルアルコール）が１ｇ含まれている。さらに、グルタミン、アラニン、グリシン、タウリン及びインスリンなどのアミノ酸構成成分がそれぞれ０．１４６ｇ、０．４５５ｇ、０．３７５ｇ、１．２５ｇ、５ｍｇほど含まれている。
ヘミカルシウムラクテートは、塩化カルシウムを除去して加えることによって、カルシウム濃度を維持し、かつエネルギー源として胚の発育が支持されるという効果がある。
また、ＰＶＡは、タンパク質の代替物質として添加するものであり、胚細胞の骨格が維持されるという効果がある。
さらに、グルタミンなどのアミノ酸は、全般的にエネルギー源として機能するとともに、タンパクの合成促進やｐＨや浸透圧の調整のため、あるいは抗酸化剤やキレート剤として働く。
具体的には、タウリンは元々卵胞・卵管に存在するタンパク質であり細胞毒を中和するキレート剤として、あるいは抗酸化剤として働くことがわかっており、この作用を利用するものである。
また、グリシンも卵管あるいは子宮に存在するタンパク質であるが、このグリシンは胚組織の浸透圧を調整する作用や胚の代謝能を向上させる機能も備えている。
インスリンも胚の代謝能を高め、胚組織におけるグルコースの吸収を促進する作用がある。
アラニンは、毒性のあるアンモニウムイオンを中和するキレート効果を備えている。
In Table 2 left column and right column, BSA means bovine serum albumin. The serum-free early embryo culture solution according to the present embodiment contains 0.54 g of hemicalcium lactate instead of calcium chloride contained in the culture solution SOF. Moreover, 1g of PVA (polyvinyl alcohol) is contained as a component contained in the serum-free early embryo culture solution which concerns on this Embodiment without being contained in other SOF. Furthermore, 0.146 g, 0.455 g, 0.375 g, 1.25 g, and 5 mg of amino acid components such as glutamine, alanine, glycine, taurine, and insulin are contained.
Hemicalcium lactate has an effect of maintaining calcium concentration and supporting embryo development as an energy source by removing calcium chloride and adding it.
PVA is added as a protein substitute and has the effect of maintaining the skeleton of the embryonic cell.
Furthermore, amino acids such as glutamine generally function as an energy source, and also work for promoting protein synthesis, adjusting pH and osmotic pressure, or as antioxidants and chelating agents.
Specifically, taurine is a protein originally present in the follicle and fallopian tube and has been known to act as a chelating agent or an antioxidant that neutralizes the cytotoxin, and utilizes this action.
Glycine is also a protein present in the oviduct or uterus, and this glycine also has an action of adjusting the osmotic pressure of embryonic tissue and a function of improving the metabolic capacity of the embryo.
Insulin also has the effect of enhancing the metabolic capacity of the embryo and promoting the absorption of glucose in the embryonic tissue.
Alanine has a chelating effect that neutralizes toxic ammonium ions.
したがって、これらのアミノ酸構成成分を無血清初期胚培養液に添加することによって、血清添加培地を用いず成分の明確な無血清培地として、しかも高価な薬剤も用いることなく、クローン胚の構築と発育を促進することができる。添加されるアミノ酸は、卵管や子宮などにゆかりのものであるため他の無血清培養液に比較すると卵管液に近い、すなわち自然に近い組成とすることができる。
また、卵丘細胞や卵管上皮細胞などとの共培養も不要であるため取扱いが容易で熟練の研究員でなくとも胚組織の培養を行うことも可能である。さらに、通常の受精胚から核移植胚などの再構築胚まで広く使用することができる。
Therefore, by adding these amino acid components to the serum-free early embryo culture medium, the construction and development of cloned embryos can be performed as a serum-free medium with a clear component without using a serum-added medium and without using expensive drugs. Can be promoted. Since the added amino acid is related to the fallopian tube, uterus, etc., it can have a composition close to that of the fallopian tube, that is, close to nature compared to other serum-free culture solutions.
In addition, since co-culture with cumulus cells, fallopian tube epithelial cells and the like is not necessary, handling is easy, and it is possible to culture embryo tissues even without skilled researchers. Furthermore, it can be widely used from normal fertilized embryos to reconstructed embryos such as nuclear transfer embryos.
次に、本実施の形態に係る無血清初期胚培養液について、受精卵の発育に係る効果を確認するための試験を実施したのでその試験方法とその結果について説明する。なお、対照区として本実施の形態に係る無血清初期胚培養液のヘミカルシウムラクテートに代えて塩化カルシウムを使用した培養液についても併せて試験を実施したので比較しながら説明する。なお、本試験に用いた実施形態に係る無血清初期胚培養液は、請求項１に記載された表に示される濃度範囲の中央値に調整されている。また、対照区の塩化カルシウムを使用した培養液は、本実施の形態に係る培養液においてヘミカルシウムラクテートに代えて塩化カルシウムを加えてヘミカルシウムラクテートの受精卵発育に関する効果を確認するために行う試験であるため、塩化カルシウムを添加した対照区にもグルタミン、アラニン、タウリン、インスリンの４種類のアミノ酸がそれぞれ、０．１４６ｇ／ｌ、０．４５５ｇ／ｌ、０．３７５ｇ／ｌ、５ｍｇ／ｌほど含まれている。
（１）試験方法
ａ）屠場未成熟卵の回収と成熟培養法
屠殺後３〜４時間以内に採取された成牛卵巣を、３０℃〜３４℃の生理食塩液で温めて実験室に運び、実験に供した。卵巣表面の卵胞（５ｍｍ〜７ｍｍ）から１８ゲージ付きのシリンジで卵母細胞を吸引採取した。
採取後、卵母細胞を抗生物質ゲンタマイシン（５０μｇ／ｍｌ）と０．３％牛血清アルブミン含有リン酸緩衝液（ＰＢＳ）で２回洗浄した。洗浄後、顆粒膜細胞の付着した卵子を回収し実験に用いた。採取された卵子／顆粒膜細胞複合体を成熟培地（ＴＣＭ１９９液、１０％胎児牛血清）で２回洗浄し、各々約３０個の卵子／顆粒膜細胞複合体を５００μｌの成熟培地に入れ、３８．５℃で５％炭酸ガス／９５％空気の加湿したインキュベーター内で２０〜２２時間培養した。
ｂ）体外受精
市販の黒毛和牛凍結***（０．５ｍｌ）を３５℃の温水中で融解し、遠心管にこの***を入れ、５ｍＭカフェインを含み牛血清アルブミンを含まないＢＯ(Brackett and Oliphant)培地（６ｍｌ）を加えて混合した。この混合液を５分間、１８００回転で遠心分離し、上澄み液を捨てた。この***洗浄操作をさらに１回同様に行った。その後血球計算盤を用いて***の濃度を２×１０６個／ｍｌに調整した。
この***液５０μｌを５ｍＭカフェイン、へパリン（１０μｇ／ｍｌ）及び脂肪酸フリーの牛血清アルブミン（６ｍｇ／ｍｌ）を含むＢＯ培地５０μｌ中に加えた。２１〜２３時間成熟培養した卵を、上記の培地で３回洗浄後、***浮遊液中に入れ、３８．５℃で５％炭酸ガス／９５％空気の加湿インキュベーター内で５時間培養することにより受精させた。
受精の時のＢＯ液中の濃度は、***１×１０６個／ｍｌ、２．５ｍＭカフェイン、へパリン（１０μｇ／ｍｌ）、牛血清アルブミン（３ｍｇ／ｍｌ）であった。
受精卵は、本実施の形態に係る無血清初期胚培養液で２回洗浄した後、毛細管様ピペットを用いて、受精卵の回りに付着している卵丘／顆粒膜細胞を裸化した。得られた裸化受精卵を、５００μｌの培地中に移した。受精卵はそれぞれの培地中で３８．５℃で５％炭酸ガス／９５％空気の加湿されたインキュベーター内で培養した。培地は、体外受精後３日目に培地交換を行い、胚の発生状況は、１２日目まで毎日顕微鏡を用いて観察し調べた。ここで、培地交換とは、胚が呼吸し、また、培地中の栄養分の代謝を行う際には、ｐＨの変動、毒性物質（エンドトキシン等）、不要物の増加等があるため、胚を除去しないように培地だけをピペットで吸って、次に新しい培地を入れることにより交換する作業をいう。
試験用培地中の組成において、ヘミカルシウムラクテートを添加した区と、塩化カルシウムを添加した区と、２区間で胚の発生を比較した。培養数は、ヘミカルシウムラクテートを添加した区では１１２、塩化カルシウムを添加した区では１１０である。
Next, since the test for confirming the effect which concerns on the growth of a fertilized egg was implemented about the serum-free early embryo culture solution which concerns on this Embodiment, the test method and its result are demonstrated. As a control group, a test was also conducted on a culture solution using calcium chloride instead of the hemicalcium lactate of the serum-free early embryo culture solution according to the present embodiment. It should be noted that the serum-free early embryo culture fluid according to the embodiment used in this test is adjusted to the median value of the concentration range shown in the table described in claim 1 . In addition, the culture solution using calcium chloride in the control group is a test performed to confirm the effect of hemicalcium lactate on fertilized egg development by adding calcium chloride in place of hemicalcium lactate in the culture solution according to the present embodiment. Therefore, the four amino acids of glutamine, alanine, taurine and insulin are also 0.146 g / l, 0.455 g / l, 0.375 g / l and 5 mg / l respectively in the control group to which calcium chloride is added. include.
(1) Test method a) Recovery of slaughterhouse immature eggs and mature culture method Adult cow ovaries collected within 3 to 4 hours after slaughtering are warmed with physiological saline at 30 ° C to 34 ° C and transported to the laboratory. It used for experiment. Oocytes were aspirated and collected from a follicle (5 mm to 7 mm) on the ovary surface with a syringe with an 18 gauge.
After collection, the oocytes were washed twice with an antibiotic gentamicin (50 μg / ml) and a phosphate buffer (PBS) containing 0.3% bovine serum albumin. After washing, the eggs with attached granulosa cells were collected and used for experiments. The collected ovum / granular membrane cell complex was washed twice with maturation medium (TCM199 solution, 10% fetal bovine serum), and about 30 ovum / granular membrane cell complexes were placed in 500 μl of maturation medium. The cells were cultured at 5 ° C. in a humidified incubator with 5% carbon dioxide gas / 95% air for 20 to 22 hours.
b) In vitro fertilization Commercial Japanese black beef frozen semen (0.5 ml) is thawed in warm water at 35 ° C., and this semen is put into a centrifuge tube and contains 5 mM caffeine and does not contain bovine serum albumin (Brackett and Oliphant) Medium (6 ml) was added and mixed. The mixture was centrifuged at 1800 rpm for 5 minutes, and the supernatant was discarded. This sperm washing operation was performed in the same manner once more. Thereafter, the sperm concentration was adjusted to 2 × 10 6 cells / ml using a hemocytometer.
50 μl of this sperm solution was added to 50 μl of BO medium containing 5 mM caffeine, heparin (10 μg / ml) and fatty acid-free bovine serum albumin (6 mg / ml). Eggs matured for 21 to 23 hours are washed three times with the above medium, placed in a sperm suspension, and cultured at 38.5 ° C. in a humidified incubator of 5% carbon dioxide / 95% air for 5 hours. Fertilized.
Concentrations in the BO solution at the time of fertilization were sperm 1 × 10 6 cells / ml, 2.5 mM caffeine, heparin (10 μg / ml), and bovine serum albumin (3 mg / ml).
After the fertilized egg was washed twice with the serum-free early embryo culture solution according to the present embodiment, cumulus / granular membrane cells adhering around the fertilized egg were naked using a capillary-like pipette. The resulting naked fertilized eggs were transferred into 500 μl of medium. Fertilized eggs were cultured in each medium at 38.5 ° C. in a humidified incubator with 5% carbon dioxide / 95% air. The medium was changed on the third day after in vitro fertilization, and the state of embryo development was observed and examined daily using a microscope until the 12th day. Here, medium exchange means that the embryo respires, and when the nutrients in the medium are metabolized, there are fluctuations in pH, toxic substances (such as endotoxin), and unwanted substances, so the embryo is removed. In order to prevent this, the medium is replaced by sucking only the medium with a pipette and then adding a new medium.
In the composition in the test medium, the development of embryos was compared between the section where hemicalcium lactate was added and the section where calcium chloride was added. The number of cultures is 112 in the group to which hemicalcium lactate was added, and 110 in the group to which calcium chloride was added.
（２）試験結果
試験の結果を表３に示す。
(2) Test results Table 3 shows the test results.
表３において、培養液の欄にヘミカルシウムラクテートとあるのは本実施の形態に係る培養液であり、塩化カルシウムとあるのは、対照区としてヘミカルシウムに代えて塩化カルシウムを添加した培養液である。表３によれば、３日目の胚分割に至った受精卵は、ヘミカルシウムラクテートの区で８１で、塩化カルシウムの区では７０と、若干ヘミカルシウムラクテートの区の方が成績はよいが、これは統計的にいえば有意な差とはいえなかった。２区間の検定は、カイ２乗検定法で行った。数値が５以下の場合にはFisherの直接確率計算法により比較した。危険率が５％以下のものを有意な差があるものと判定した。
一方、８日目に桑実胚あるいは胚盤胞まで至っている受精卵は、ヘミカルシウムラクテートの区では符号ａで示すとおり５４であるのに対し、塩化カルシウムの区では符号ｂで示すとおり３３となった。この結果については、前述のとおり５％の危険率でカイ２乗検定を実施した結果、ａ，ｂの異符号間で有意な差と認められた。但し、胚盤胞のみで見るとヘミカルシウムラクテートの区で１０であるのに対して塩化カルシウムの区では５であり、これに対しては試料数が小さいために統計的には有意な差は認められなかったが、胚盤胞に至る確率もほぼ倍の数値を得ることができた。
以上の試験結果からすると、塩化カルシウムよりもヘミカルシウムラクテートを添加した無血清初期胚培養液の方が受精卵が発育する際、特に８日目以降の桑実胚から胚盤胞に至るまでの培養に大きな効果を発揮しており、培養液として望ましいことがわかる。
なお、本試験を実施するに際して、本実施の形態に係る無血清初期胚培養液を調製する際に各成分の液量を測定しながら行ったが、測定には略１０％の誤差が含まれていたものの、その試験結果については概ね同等の結果が得られた。
In Table 3, in the column of the culture solution, hemicalcium lactate is the culture solution according to the present embodiment, and calcium chloride is a culture solution in which calcium chloride is added instead of hemicalcium as a control group. is there. According to Table 3, the fertilized egg that reached the day 3 embryo splitting was 81 in the hemicalcium lactate group, 70 in the calcium chloride group, and the hemicalcium lactate group had better results, This was not a statistically significant difference. The test for two sections was performed by the chi-square test method. When the numerical value was 5 or less, comparison was made by Fisher's direct probability calculation method. A risk rate of 5% or less was determined to have a significant difference.
On the other hand, the fertilized egg reaching the morula or blastocyst on the 8th day is 54 as indicated by the symbol a in the hemicalcium lactate group, whereas it is 33 as indicated by the symbol b in the calcium chloride group. became. As described above, the chi-square test was performed at a risk rate of 5% as described above. As a result, it was recognized that there was a significant difference between the a and b different signs. However, in the blastocyst alone, it is 10 in the hemi-calcium lactate group, whereas it is 5 in the calcium chloride group. Although not recognized, the probability of reaching a blastocyst was almost doubled.
From the above test results, when the fertilized egg develops in the serum-free early embryo culture solution to which hemicalcium lactate is added rather than calcium chloride, especially from the morula to the blastocyst after the 8th day. It shows a great effect on the culture, which is desirable as a culture solution.
In carrying out this test, the serum-free early embryo culture solution according to this embodiment was prepared while measuring the amount of each component, but the measurement includes an error of approximately 10%. However, the test results were almost equivalent.
次に、本実施の形態に係る無血清初期胚培養液に対し、核移植した胚組織の培養に関する効果について他の無血清培養液と比較する試験を実施したのでその結果について説明する。ここでも、本実施の形態に係る無血清初期胚培養液とは、請求項１に記載された無血清初期胚培養液の中央値の成分を備える培養液である。
（１）試験方法
ａ）屠場未成熟卵の回収と成熟培養法
屠場未成熟卵を、ヘミカルシウムラクテートの効果を確認するための試験と同じ方法で回収し、成熟培養した。
ｂ）卵子の除核
成熟培養後、卵子を０．１％ヒアルロニダーゼ添加ＴＣＭ１９９液内で卵子に付着している卵丘／顆粒膜細胞を裸化した。これらの裸化卵子から第１極体を放出している成熟卵子を実体顕微鏡下で選別し、５μｇ／ｍｌヘキスト添加ＴＣＭ１９９液内で１０分間染色した。その後、倒立顕微鏡下でマイクロマニピュレーターに取り付けたガラス微細針を使って卵子の透明体を切開して第１極体と周辺の細胞質を押し出し、紫外線をあてて押し出した卵子細胞質内の核を確認した。
ｃ）細胞融合
雌成牛の皮膚に由来する培養細胞株をトリプシン処置してドナー核となる体細胞を準備した。これらの体細胞を上記の方法で除核した卵子の囲卵腔内に微細ガラスピペットを使って注入し卵子細胞質と密着させた。細胞注入後、卵子を細胞融合液（０．３Ｍマンニトール、０．０５ｍＭカルシウム、０．１ｍＭマグネシウム）内に移し、２．２５ｋＶ／ｃｍの直流電流を２５μｓｅｃ間隔で２回与え、卵子細胞質と注入した体細胞を電気的に融合した。その後、細胞融合した胚を１０μｇ／ｍｌシクロヘキシミド添加ＴＣＭ１９９液内で４時間培養して活性化処置した。
活性化処置後に得られた核移植胚を本実施の形態に係る無血清初期胚培養液で３回洗浄し、１００μｌの培地中に移した。核移植胚はそれぞれの培地中で３８．５℃で５％炭酸ガス／９５％空気の加湿されたインキュベーター内で培養した。培地は、体外受精後３日目に培地交換を行い、胚の発生状況は、１２日目まで毎日顕微鏡を用いて観察し調べた。
試験では、核移植胚を本実施の形態に係る無血清初期胚培養液で培養した区と５％牛胎仔血清（ＦＢＳ：Fetal Bovine Serum）添加ＣＲ１ａａ液で培養した区の２区間で胚の発生を比較した。培養数は、本実施の形態に係る無血清初期胚培養液による区では１０４、ＦＢＳを含む培地による区は１５４である。
Next, since the test which compares with the serum-free early embryo culture solution which concerns on this Embodiment about the effect regarding the culture | cultivation of the embryo-transplanted embryo tissue with another serum-free culture solution is demonstrated. Again, the serum-free early embryo culture solution according to the present embodiment is a culture solution comprising the median component of the serum-free early embryo culture solution according to claim 1 .
(1) Test method a) Recovery of slaughterhouse immature egg and maturation culture method Slaughterhouse immature egg was recovered by the same method as the test for confirming the effect of hemicalcium lactate and matured.
b) Enucleation of ovum After maturation culture, cumulus / granular membrane cells adhering to the ovum were naked in the TCM199 solution supplemented with 0.1% hyaluronidase. Mature eggs releasing the first polar body from these naked eggs were selected under a stereomicroscope and stained for 10 minutes in a TCM199 solution containing 5 μg / ml Hoechst. Then, using a glass microneedle attached to a micromanipulator under an inverted microscope, the transparent body of the ovum was incised to extrude the first polar body and the surrounding cytoplasm, and the nucleus in the ooplasm that was extruded by applying ultraviolet light was confirmed. .
c) Cell fusion A cultured cell line derived from the skin of an adult female cow was treated with trypsin to prepare somatic cells as donor nuclei. These somatic cells were injected into the surrounding space of the ovum enucleated by the above method using a fine glass pipette and brought into close contact with the ovum cytoplasm. After cell injection, the ovum was transferred into a cell fusion solution (0.3 M mannitol, 0.05 mM calcium, 0.1 mM magnesium), and a direct current of 2.25 kV / cm was applied twice at 25 μsec intervals and injected with the ovum cytoplasm. Somatic cells were fused electrically. Thereafter, the cell-fused embryo was cultured in TCM199 solution supplemented with 10 μg / ml cycloheximide for 4 hours for activation treatment.
Nuclear transfer embryos obtained after the activation treatment were washed three times with the serum-free early embryo culture medium according to the present embodiment and transferred into 100 μl of medium. Nuclear transfer embryos were cultured in each medium at 38.5 ° C. in a humidified incubator with 5% carbon dioxide / 95% air. The medium was changed on the third day after in vitro fertilization, and the state of embryo development was observed and examined daily using a microscope until the 12th day.
In the test, embryo development occurred in two sections: a section in which the nuclear transfer embryo was cultured in the serum-free early embryo culture medium according to the present embodiment and a section in which the 5% fetal bovine serum (FBS) -added CR1aa solution was cultured. Compared. The number of cultures is 104 for the serum-free early embryo culture medium according to the present embodiment, and 154 for the medium containing FBS.
（２）試験結果
試験の結果を表４に示す。
(2) Test results Table 4 shows the test results.
表４によれば、２日目において胚分割に至っている核移植胚の数は、本実施の形態に係る無血清初期胚培養液の区で９３、ＦＢＳを含む培地による区では１４０となっており、発育における差はほとんど見られない。もちろん、検定によって有意な差は認められなかった。
しかし、７日目において桑実胚あるいは胚盤胞まで至っている核移植胚は、本実施の形態に係る無血清初期胚培養液による区では６１、ＦＢＳを含む培地による区では７７と、桑実胚と胚盤胞の数を加えた場合では統計的な有意差はないものの若干ながら本実施の形態に係る無血清初期胚培養液による区の方が発育の促進効果がより高く現れている。
但し、胚盤胞のみに限定してみると、本実施の形態に係る無血清初期胚培養液にのよる区が符号ａで示されるように１８であるのに対し、ＦＢＳを含む培地の方が符号ｂで示されるように５８と２倍以上の確率で発育していることがわかる。この数については、前述の５％の危険率でカイ２乗検定を実施したところ、ａ，ｂの異符号間で有意な差として認められた。
以上の試験結果から、通常クローン胚の培養に使用されているＣＲ１ａａ液と対照させながら、本実施の形態に係る無血清初期胚培養液が体細胞クローン胚の発育を支持するか否かについては、桑実期胚以上への核移植胚の発育は血清を添加しているＣＲ１ａａ液と同等であった。また、胚盤胞までの発育では、ＣＲ１ａａ液と有意な差を生じてしまったが、本実施の形態に係る無血清初期胚培養液であっても、ＣＲ１ａａ液の約５割の確率では培養が可能であることが示された。
本試験を実施する際においても、無血清初期胚培養液を調製する際に、各成分の液量を測定しながら行ったが、測定には略１０％の誤差が含まれていたものの、その試験結果については概ね同等の結果が得られた。
According to Table 4, the number of nuclear transfer embryos that reached embryo division on the second day was 93 in the group of the serum-free early embryo culture solution according to the present embodiment, and 140 in the group with the medium containing FBS. There is almost no difference in development. Of course, no significant difference was found by the test.
However, the nuclear transfer embryos that reached the morula or blastocyst on the seventh day were 61 in the group using the serum-free early embryo culture solution according to the present embodiment, 77 in the group using the medium containing FBS, When the number of embryos and blastocysts is added, there is no statistically significant difference, but the growth-promoting effect is more apparent in the group with the serum-free early embryo culture medium according to the present embodiment.
However, when limited to blastocysts only, the group according to the serum-free early embryo culture medium according to the present embodiment is 18 as indicated by the symbol a, whereas the medium containing FBS As shown by the symbol b, it can be seen that it grows with a probability of 58 or more than twice. About this number, when the chi-square test was carried out at the above-mentioned 5% risk rate, it was recognized as a significant difference between the different signs of a and b.
From the above test results, whether or not the serum-free early embryo culture medium according to the present embodiment supports the development of somatic clone embryos while contrasting with the CR1aa liquid normally used for culturing cloned embryos. The development of nuclear transfer embryos beyond the morula stage was similar to that of CR1aa solution supplemented with serum. Further, in the development up to the blastocyst, there was a significant difference from the CR1aa solution, but even with the serum-free early embryo culture solution according to the present embodiment, the culture is performed with a probability of about 50% of the CR1aa solution. Was shown to be possible.
In conducting this test, the serum-free early embryo culture solution was prepared while measuring the liquid amount of each component. Although the measurement contained an error of about 10%, The test results were almost equivalent.
本発明に係る無血清初期胚培養液は、組成が明確であると同時に安価な成分を用いるものであるので、低いコストで量産が可能であり、体外受精卵の培養液の他、クローン胚の構築、発育のための培養液として利用することができる。
Since the serum-free early embryo culture medium according to the present invention uses a low-cost component at the same time as its composition is clear, it can be mass-produced at a low cost. It can be used as a culture solution for construction and development.
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