JP2021529505A - イヌ科動物の卵子をガラス化凍結し解凍する方法およびそれによる凍結解凍卵子 - Google Patents
イヌ科動物の卵子をガラス化凍結し解凍する方法およびそれによる凍結解凍卵子 Download PDFInfo
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Abstract
Description
イヌ科動物の成熟卵子を採取する過程(1過程)、
前記採取した卵子をガラス化凍結過程(2過程)を行い、
混合液、平衡溶液、ガラス化溶液を準備する過程(2過程−1)、
2過程‐1において、前記混合液は10〜30% SSS(Serum Substitute Supplement)100重量部にmHTF(Modified HTF Medium−HEPES with Gentamicin)300〜1000重量部を混合したものであり、
前記平衡溶液は6.5〜8.5%(v/v)エチレングリコール(Ethylene glycol)、6.5〜8.5%(v/v)ジメチルスルホキシド(Dimethyl sulfoxide)、10〜30%デキストラン血清補充剤(Dextran Serum Supplement)そして25〜45μg/mlの硫酸ゲンタマイシン(gentamicin sulfate)を含むHEPES緩衝溶液から構成され、
前記ガラス化溶液(Vitrification Solution)は5〜25%(v/v)エチレングリコール(Ethylene glycol)、5〜25%(v/v)ジメチルスルホキシド(Dimethyl sulfoxide)、10〜30%デキストラン血清補充剤(Dextran Serum Supplement)、0.3〜0.6Mスクロース(Sucrose)、25〜45μg/mlの硫酸ゲンタマイシン(gentamicin sulfate)を含むHEPES緩衝溶液から構成され、
前記回収した卵子の卵丘細胞の除去過程を経る(denuding)過程(2過程−2)、
前記卵丘細胞を除去した成熟卵子を室温に平衡させた混合液に処理する過程(2過程−3)、
前記卵子が含まれている混合液に平衡溶液を追加して処理した後、再び平衡溶液をさらに追加して処理する過程(2過程−4)、
前記処理を経た卵子を再び平衡溶液で処理する過程(2過程−5)、
処理した卵子をガラス化溶液に移して処理する過程(2過程−6)、および
卵子を卵子凍結道具に載せてガラス化溶液の除去後に直ちに液体窒素に浸漬して保管する過程(2過程−7)、
を含むイヌ科動物の卵子をガラス化凍結し解凍する方法を提供する。
解凍溶液、希釈溶液、洗浄溶液、回復培養液を準備する過程を行うが(3過程−1)、
3過程―1において、解凍溶液(Thawing Solution)は0.5〜1.5Mスクロース(Sucrose)、10〜30%デキストラン血清補充剤(Dextran Serum Supplement)そして25〜45μg/mlの硫酸ゲンタマイシン(gentamicin sulfate)を含むHEPES緩衝溶液から構成され、
希釈溶液(Dilution Solution)は0.3〜0.7Mスクロース(Sucrose)、10〜30%デキストラン血清補充剤(Dextran Serum Supplement)そして25〜45μg/mlの硫酸ゲンタマイシン(gentamicin sulfate)を含むHEPES緩衝溶液から構成され、
回復培養液(Recovery Medium)はHEPES緩衝溶液、ピルビン酸ナトリウム(Sodium pyruvate)、硫酸ゲンタマイシン(gentamicin sulfate)、FBS(Fetal Bovine Serum)を混合したものから構成され、
解凍溶液(Thawing Solution)をインキュベータで35〜40℃に加温させる過程(3過程−2)、
回復培養液(RecoveryMedium)をインキュベータで35〜40℃に加温させる過程(3過程−3)、
液体窒素に保管された卵子凍結道具(CryoTop)を取り出して加温した解凍溶液に入れ、卵子凍結道具(CryoTop)に付着している卵子を分離して処理する過程(3過程−4)、
前記分離した卵子を常温に平衡させた希釈溶液に移して処理する過程(3過程−5)、
前記処理された卵子を常温に平衡させた2個の洗浄溶液に移して順次処理する過程(3過程−6)、および
前記過程後、回復培養のために前記加温した回復培養液で培養する過程(3過程−7)、
を含むことを特徴とするイヌ科動物の卵子をガラス化凍結し解凍する方法を提供する。
Claims (3)
- イヌ科動物の成熟卵子を採取する過程(1過程)、
前記採取した卵子に対してガラス化凍結過程(2過程)を行い、
混合液、平衡溶液、ガラス化溶液を準備する過程(2過程−1)、
前記回収した卵子の卵丘細胞を除去する過程(denuding)(2過程−2)、
前記卵丘細胞を除去した成熟卵子を室温に平衡させた混合液に処理する過程(2過程−3)、
前記卵子が含まれている混合液に平衡溶液を追加して処理した後、再び平衡溶液をさらに追加して処理する過程(2過程−4)、
前記処理を経た卵子を再び平衡溶液で処理する過程(2過程−5)、
処理した卵子をガラス化溶液に移して処理する過程(2過程−6)、および
卵子を卵子凍結道具に載せてガラス化溶液の除去後に直ちに液体窒素に浸漬して保管する過程(2過程−7)
を含むことを特徴とする、イヌ科動物の卵子をガラス化凍結し解凍する方法。 - ガラス化凍結卵子に対して融解する過程(3過程)を行い、
解凍溶液、希釈溶液、洗浄溶液、回復培養液を準備する過程(3過程−1)、
解凍溶液(Thawing Solution)をインキュベータで35〜40℃に加温させる過程(3過程−2)、
回復培養液(Recovery Medium)をインキュベータで35〜40℃に加温させる過程(3過程−3)、
液体窒素に保管された卵子凍結道具(CryoTop)を取り出して加温した解凍溶液に入れ、卵子凍結道具に付着している卵子を分離して処理する過程(3過程−4)、
前記分離した卵子を常温に平衡させた希釈溶液に移して処理する過程(3過程−5)、
前記処理された卵子を常温に平衡させた2個の洗浄溶液に移して順次処理する過程(3過程−6)、および
前記過程後、回復培養をために前記前培養した回復培養液で培養する過程(3過程−7)
を含むことを特徴とする、請求項1に記載のイヌ科動物の卵子をガラス化凍結し解凍する方法。 - 請求項1または2に記載の方法により形成された凍結解凍卵子。
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