JP3044323B1 - Cell cryopreservation method - Google Patents
Cell cryopreservation methodInfo
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- JP3044323B1 JP3044323B1 JP11001975A JP197599A JP3044323B1 JP 3044323 B1 JP3044323 B1 JP 3044323B1 JP 11001975 A JP11001975 A JP 11001975A JP 197599 A JP197599 A JP 197599A JP 3044323 B1 JP3044323 B1 JP 3044323B1
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Abstract
【要約】
【課題】 細胞の凍結保存方法の提供。
【解決手段】 細胞を、ガラス化液の存在下で液体窒素
中に滴下することを特徴とする細胞の凍結保存方法。A method for cryopreserving cells is provided. SOLUTION: The method for cryopreservation of cells is characterized by dropping cells into liquid nitrogen in the presence of a vitrification solution.
Description
【０００１】[0001]
【発明の属する技術分野】本発明は細胞の凍結保存方法
に関する。The present invention relates to a method for cryopreserving cells.
【０００２】[0002]
【従来の技術】細胞の凍結保存は、細胞を低濃度のグリ
セロールやジメチルスルフォオキサイドなどの凍結防御
剤で平衡させ、ガラスアンプルやプラスティックストロ
ー等の容器に充填し、徐々に温度を下げていくことによ
って行う緩慢冷却法、又は細胞を高濃度の凍結防御剤と
ともに容器に入れ、急激に液体窒素温度まで下げて保存
するガラス化法によって行われてきた。2. Description of the Related Art In cryopreservation of cells, cells are equilibrated with a low concentration of a cryoprotectant such as glycerol or dimethyl sulfoxide, filled in a container such as a glass ampule or a plastic straw, and the temperature is gradually lowered. Or a vitrification method in which cells are placed in a container with a high concentration of a cryoprotectant and rapidly cooled to the temperature of liquid nitrogen and stored.
【０００３】緩慢冷却法は、低濃度の凍結防御剤を用い
るため、細胞に対する化学的毒性や物理的ストレスが比
較的低く凍結操作も容易であるという利点があるため、
一般的な培養細胞などに広く用いられている方法であ
る。しかしながら、緩慢冷却法では、細胞外液に氷晶を
形成させることによって細胞内を脱水していくことか
ら、氷晶によって細胞がダメージを受ける、冷却速度を
コントロールするための機器が必要である、低温域を徐
々に通過するので低温感受性の高い細胞の保存には向か
ないなどの問題点があり、牛未受精卵子などの凍結保存
には適していなかった。[0003] The slow cooling method has an advantage that the chemical toxicity and physical stress on cells are relatively low and the freezing operation is easy because a low concentration of the cryoprotectant is used.
This method is widely used for general cultured cells and the like. However, in the slow cooling method, since the inside of the cell is dehydrated by forming ice crystals in the extracellular solution, the cells are damaged by the ice crystals, and a device for controlling the cooling rate is required. Since the cells gradually pass through the low-temperature region, they are not suitable for preserving cells having high low-temperature sensitivity, and thus are not suitable for cryopreservation of unfertilized bovine eggs.
【０００４】ガラス化保存法は、細胞を高濃度の溶液と
ともに容器に充填し、急激に液体窒素温度まで冷却する
ことによって、細胞内外ともに氷晶を形成しないガラス
化状態にして保存する方法である。この方法は、氷晶に
よる細胞へのダメージがない、低温域を素早く通過する
ので低温感受性が高い細胞へも適用できる、特別な機器
を必要としないなどの利点から近年注目を浴びている方
法である。しかしながら、ガラス化保存は非常に高濃度
の凍結防御剤を含んだガラス化液を用いなければならな
いため、細胞をガラス化液に添加し、又はガラス化液を
除去した場合にその浸透圧差によって物理的なショック
を受ける、高濃度の溶液による化学的毒性によって細胞
が死んでしまうなどの問題点がある。[0004] The vitrification preservation method is a method in which cells are packed in a container together with a high-concentration solution, and rapidly cooled to the temperature of liquid nitrogen, whereby the cells are stored in a vitrified state in which ice crystals are not formed inside and outside the cells. . This method has attracted attention in recent years because of the advantages that it does not damage cells due to ice crystals, can be applied to cells sensitive to low temperatures because it passes through the low temperature range quickly, and does not require special equipment. is there. However, vitrification requires the use of a vitrification solution containing a very high concentration of a cryoprotectant, so if cells are added to the vitrification solution or the vitrification solution is removed, the physical difference due to the osmotic pressure difference occurs. There is a problem that cells are killed due to a chemical shock due to chemical shock and chemical toxicity due to a highly concentrated solution.
【０００５】さらに、従来のガラス化法では細胞をガラ
ス化液とともにガラスアンプル、クライオチューブある
いはプラスティックストローといった容器に充填してか
ら液体窒素中に投入して冷却を行うため、その分冷却速
度が遅延していた。Furthermore, in the conventional vitrification method, cells are charged together with a vitrification liquid into a container such as a glass ampule, a cryotube or a plastic straw, and then charged into liquid nitrogen for cooling. Was.
【０００６】[0006]
【発明が解決しようとする課題】本発明は、細胞の凍結
保存方法を提供することを目的とする。An object of the present invention is to provide a method for cryopreserving cells.
【課題を解決するための手段】本発明者は、上記課題を
解決するため鋭意研究を行った結果、細胞を少量のガラ
ス化液存在下で、容器に充填することなく直接液体窒素
中へ滴下することによって、細胞を凍結保存し得ること
を見出し、本発明を完成するに至った。すなわち、本発
明は、細胞を、ガラス化液の存在下で液体窒素中に滴下
することを特徴とする細胞の凍結保存方法である。細胞
としては、例えば動物又はヒトの生殖細胞又は体細胞が
挙げられる。以下、本発明を詳細に説明する。Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, in the presence of a small amount of vitrification liquid, the cells were dropped directly into liquid nitrogen without filling the container. As a result, they found that cells could be cryopreserved, and completed the present invention. That is, the present invention is a method for cryopreserving cells, which comprises dropping cells into liquid nitrogen in the presence of a vitrification liquid. Cells include, for example, animal or human germ cells or somatic cells. Hereinafter, the present invention will be described in detail.
【０００７】[0007]
【発明の実施の形態】本発明の凍結保存方法は、細胞を
含むガラス化液（液滴）を直接液体窒素中へ滴下するこ
と、すなわち、当該ガラス化液を容器に充填せずに、ガ
ラス化液が液体窒素に直接触れるようにして冷却を行う
ことを特徴とするものであり、マイクロドロップレット
法又はガラス化保存法と呼ぶことができる。本発明のガ
ラス化保存法で保存される細胞としては、特に限定され
るものではなく、例えば、牛、山羊、羊、豚、マウス、
ラット、ウサギ等の動物（実験動物、家畜等）の生殖細
胞及び体細胞、又はヒトの生殖細胞及び体細胞などが例
示される。BEST MODE FOR CARRYING OUT THE INVENTION The cryopreservation method of the present invention comprises dropping a vitrification liquid (droplets) containing cells directly into liquid nitrogen. It is characterized in that cooling is performed by bringing the liquefied liquid into direct contact with liquid nitrogen, and can be called a microdroplet method or a vitrification storage method. Cells preserved by the vitrification preservation method of the present invention are not particularly limited, and include, for example, cows, goats, sheep, pigs, mice,
Examples include germ cells and somatic cells of animals such as rats and rabbits (experimental animals, domestic animals, etc.), and germ cells and somatic cells of humans.
【０００８】上記細胞は、通常の手法により採取するこ
とができる。例えば、生殖細胞を採取する場合は、牛の
未受精卵子については、卵巣の表面にある卵胞より注射
器により吸引することによって採取することができる。
胚については、未受精卵子と精子とを体外受精させ、そ
の後培養することによって得ることができる。あるい
は、ホルモンにより過剰排卵処理をした動物の子宮を外
科的又は非外科的に灌流することによって採取すること
もできる。なお、生殖細胞とは、未受精卵子、精子又は
受精後０〜10日目（1〜200細胞期）における細胞若しく
は胚を意味する。本発明においては、特に従来の方法で
は保存が困難である牛の未受精卵子若しくは卵割期胚、
豚の未受精卵子若しくは胚、又はマウスの未受精卵子な
どが好適に使用される。The above cells can be collected by a usual technique. For example, when collecting germ cells, unfertilized bovine ova can be collected by aspirating a follicle on the surface of the ovary with a syringe.
Embryos can be obtained by in vitro fertilization of unfertilized eggs and sperm, followed by culturing. Alternatively, the uterus of an animal that has been superovulated with a hormone can be collected by surgically or non-surgically perfusing the uterus. In addition, a germ cell means an unfertilized egg, a sperm, or a cell or embryo at 0 to 10 days after fertilization (1-200 cell stage). In the present invention, in particular, unfertilized eggs or cleavage embryos of cattle that are difficult to preserve by conventional methods,
An unfertilized egg or embryo of a pig, or an unfertilized egg of a mouse is preferably used.
【０００９】また、体細胞を採取する場合は、例えば皮
膚細胞であれば、動物より皮膚を採取し、コラーゲンな
どの酵素で組織を分散させることによって採取できる。
得られた細胞を適当な細胞培養液（例えばウシ胎児血清
を含むH-TCM199、RPMI1640又はDMEMなど）又は緩衝液
（PBS等）に添加した後、ガラス化液に浸漬する。ガラ
ス化液とは、低温域における液体の氷晶形成を抑制する
凍結防御剤、及び細胞の脱水を促し細胞膜を保護する働
きのある糖などを含み、液体窒素中でも氷晶を形成しな
いのに十分な濃度を持った溶液を意味し、通常は5.5Mエ
チレングリコール及び１Mシュクロースの混合物、又は4
0%エチレングリコール、18%フィコール及び0.3Mトレハ
ロースの混合物が使用される。[0009] When collecting somatic cells, for example, skin cells can be collected by collecting skin from an animal and dispersing the tissue with an enzyme such as collagen.
The obtained cells are added to a suitable cell culture solution (for example, H-TCM199, RPMI1640 or DMEM containing fetal bovine serum) or a buffer (PBS or the like), and then immersed in a vitrification solution. The vitrification liquid includes a cryoprotectant that suppresses the formation of ice crystals in the liquid at low temperatures, and a sugar that promotes dehydration of cells and protects cell membranes, and is sufficient to prevent the formation of ice crystals even in liquid nitrogen. Solution having a high concentration, usually a mixture of 5.5M ethylene glycol and 1M sucrose, or 4
A mixture of 0% ethylene glycol, 18% ficoll and 0.3M trehalose is used.
【００１０】次に、細胞を含有する少量のガラス化液を
ピペットで吸い取り、容器に入れることなく液体窒素中
に直に滴下する。「容器に入れることなく」とは、容器
に充填した細胞含有ガラス化液を液体窒素中に入れるこ
とを意味するのではなく、細胞を含有するガラス化液を
直接液体窒素に接触させることを意味する。この場合、
液体窒素を含むタンク中に細胞含有ガラス化液を滴下す
ることもできるが、細胞の保存性を良くし、また、どの
細胞であるかを識別することができるようにするため、
液体窒素を充填した試験管又はクライオチューブ（細胞
の種類や日付等をラベルしたものが好ましい）の中に細
胞含有ガラス化液を滴下して凍結させ、そのチューブを
液体窒素タンクに改めて保存することが好ましい。ま
た、「少量の」とは、液体窒素に滴下したときに瞬時に
細胞を凍結させることができる程度の量を意味し、具体
的には1〜10μl、好ましくは4〜8μlを意味する。な
お、かかる少量のガラス化液中に含まれる細胞数は、1
μlあたり1〜1000万個、好ましくは1〜10万個である。
凍結保存後の融解は、通常の手法（例えば37℃）により
行うことができる。その後、通常の細胞培養培地を用
い、37〜39℃、5%CO2存在下で培養し、発生・分化を行
わせる。Next, a small amount of the vitrification liquid containing the cells is sucked up with a pipette and directly dropped into liquid nitrogen without being put in a container. "Without placing in a container" does not mean that the cell-containing vitrification solution filled in the container is placed in liquid nitrogen, but that the cell-containing vitrification solution is directly contacted with liquid nitrogen. I do. in this case,
Cell-containing vitrification liquid can also be dropped into a tank containing liquid nitrogen, but in order to improve the preservability of cells and to be able to identify which cells they are,
Drop the cell-containing vitrification liquid into a test tube or cryotube filled with liquid nitrogen (preferably labeled with cell type and date), freeze it, and store the tube in a liquid nitrogen tank again. Is preferred. The term "small amount" means an amount capable of instantaneously freezing cells when dropped into liquid nitrogen, and specifically 1 to 10 µl, preferably 4 to 8 µl. The number of cells contained in such a small amount of vitrification liquid is 1
It is 10 to 10 million, preferably 1 to 100,000 per μl.
Thawing after cryopreservation can be performed by a usual method (for example, 37 ° C.). Thereafter, the cells are cultured in a normal cell culture medium at 37 to 39 ° C. in the presence of 5% CO 2 to cause development and differentiation.
【００１１】[0011]
【実施例】以下、実施例により本発明をさらに詳細に説
明する。但し、本発明はこれら実施例にその技術的範囲
が限定されるものではない。 〔実施例1〕牛体外成熟卵子の保存 1．供試材料 本実施例では、従来のガラス化保存法では保存が困難で
あり、融解したときの再発生率が低いとされている牛体
外成熟卵子を用いた。The present invention will be described in more detail with reference to the following examples. However, the technical scope of the present invention is not limited to these examples. [Example 1] Preservation of cow extracorporeal mature egg Test Materials In this example, extracorporeal bovine eggs, which are difficult to preserve by the conventional vitrification preservation method and whose regrowth rate upon melting is low, were used.
【００１２】2．牛体外成熟卵子の作出 食肉処理場由来の牛卵巣より卵子を採取し、H-TCM199
（GIBCO社）、5%ウシ胎児血清（FCS）、 FSH（卵胞刺激
ホルモン；デンカ製薬社）及びE2（エストラディオール
17-β；SIGMA社）を含有する溶液中で38.5℃、5%CO2の
条件下で21〜24時間体外成熟させた後、卵丘細胞を除去
した。2. Production of extracorporeal matured eggs Eggs were collected from bovine ovaries from slaughterhouses, and H-TCM199
(GIBCO), 5% fetal calf serum (FCS), FSH (follicle stimulating hormone; Denka Pharmaceutical) and E2 (estradiol)
After in vitro maturation for 21 to 24 hours under a condition of 38.5 ° C. and 5% CO 2 in a solution containing 17-β (SIGMA), cumulus cells were removed.
【００１３】3．ガラス化保存法 上記卵子を、3％のエチレングリコール溶液に12〜15分
間平衡させた後、ガラス化液（5.5Mエチレングリコール
+1Mシュークロス）に30秒間浸し、ガラスピペットを用
いて卵子を少量の液（4-8μl）とともに液体窒素中に滴
下した。それらの小滴をクライオチューブに入れて1〜7
日保存した。融解及びガラス化液の除去は、小滴を20％
ウシ胎仔血清加199培養液中に添加することによって行
った。3. Vitrification preservation method The above eggs were equilibrated in a 3% ethylene glycol solution for 12 to 15 minutes, and then vitrified (5.5 M ethylene glycol).
+ 1M shoe cloth) for 30 seconds, and the eggs were dropped into liquid nitrogen together with a small amount of liquid (4-8 μl) using a glass pipette. Put those droplets into a cryotube and put them in 1-7
Days saved. Melting and removal of vitrification liquid, droplets 20%
It was carried out by adding to 199 culture medium supplemented with fetal bovine serum.
【００１４】ストロー内でのガラス化保存は、卵子を10
％のエチレングリコール液に10分間平衡させ、ガラス化
液（40％エチレングリコール+18％フィコール+0.3Ｍト
レハロース）に浸漬してからストロー内に充填し、２分
後に液体窒素中に投入し、1〜7日保存した。融解及びガ
ラス化液の除去は、ストローを空気中で10秒間保持して
から20℃の水中で融解し、卵子を0.5 Mトレハロース液
中に取り出し、10分間保持してガラス化液を除去した。
なお、何ら凍結処理を施さない卵子を対照区とした。[0014] Vitrification storage in a straw, 10 eggs
% Ethylene glycol solution for 10 minutes, immersed in a vitrification solution (40% ethylene glycol + 18% ficoll + 0.3 M trehalose), filled in a straw, and after 2 minutes, poured into liquid nitrogen. Saved ~ 7 days. For the melting and removal of the vitrification liquid, the straw was kept in the air for 10 seconds, then thawed in water at 20 ° C., the ovum was taken out in a 0.5 M trehalose liquid, and kept for 10 minutes to remove the vitrification liquid.
Eggs not subjected to any freezing treatment were used as control groups.
【００１５】4．生存性の判定 融解した卵子を体外受精し、その後の発生率を観察する
ことによって生存性の判定の指標とした。4. Determination of viability Melted ova were fertilized in vitro, and the incidence was observed to determine the survival.
【００１６】5．結果 本発明のマイクロドロップレット法によってガラス化保
存した牛体外成熟卵子は、体外受精後75.9％（41／54）
が分割し、29.6％（16／54）が胚盤胞へ発生した（表
１）。対照区の卵子の発生率は、受精後分割した細胞に
ついては88.7％（47／53）、胚盤胞へ発生した細胞につ
いては41.5％（22／53）であり、ガラス化卵子の成績と
統計的に有意な差は認められなかった。従って、ガラス
化卵子は、融解後であっても対照区と同様の生存性を有
するものであると判断することができる。また、ストロ
ー内でガラス化保存した卵子の発生率は、受精後分割し
た細胞については14.8％（12／81）、胚盤胞へ発生した
細胞については1.2％（1／81）であり、マイクロドロッ
プレット法よりも低い値であった。（表１）。従って、
本発明の方法（マイクロドロップレット法）は、従来法
（ストロー法）と比較して牛体外成熟卵子に対する保存
性及び融解後の生存性に優れていることが確認された。[0016] 5. Results 75.9% (41/54) of in vitro matured bovine eggs stored vitrified by the microdroplet method of the present invention after in vitro fertilization
, And 29.6% (16/54) developed into blastocysts (Table 1). The incidence of eggs in the control group was 88.7% (47/53) for cells divided after fertilization, and 41.5% (22/53) for cells that developed into blastocysts. No significant difference was observed. Therefore, it can be determined that the vitrified ovum has the same viability as the control group even after being thawed. In addition, the incidence of eggs vitrified and stored in a straw was 14.8% (12/81) for cells divided after fertilization, and 1.2% (1/81) for cells that developed into blastocysts. The value was lower than the droplet method. (Table 1). Therefore,
It was confirmed that the method of the present invention (microdroplet method) is superior to the conventional method (straw method) in the preservation of the extracorporeally matured egg and the survival after thawing.
【００１７】 [0017]
【００１８】〔実施例2〕牛の体外受精３日目胚の保存 1．供試材料 本実施例では、従来のガラス化法では保存が比較的難し
いとされている、牛体外受精３日目胚（体外受精日=0
日、8-16細胞期）を試験に供した。Example 2 Preservation of Embryo on Day 3 of In Vitro Fertilization of Cattle Test Material In this example, it was considered that preservation was relatively difficult with the conventional vitrification method.
Day, 8-16 cell stage).
【００１９】2．胚の作出 食肉処理場由来の牛卵巣より採取した卵子を、常法によ
り体外成熟・体外受精させ、CR1aa及びリノール酸アル
ブミンを含む溶液中で、38.5℃、5%O2、5%CO2、90%N2の
条件下で３日間培養して、８〜16細胞期に発生した胚を
試験に用いた。2. Oocytes collected from bovine ovaries from the slaughterhouse, in vitro maturation and in vitro fertilization by a conventional method, in a solution containing CR1aa and albumin linoleate, 38.5 ℃, 5% O 2 , 5% CO 2 , Embryos cultured at 90% N 2 for 3 days and developed at the 8-16 cell stage were used for the test.
【００２０】3．ガラス化保存法 胚を2％のエチレングリコール液に12〜16分間平衡さ
せ、ガラス化液に60秒間浸し、液体窒素中に滴下した。
また、一部の胚は0.25ccのプラスティックストローに充
填してから液体窒素中に投入した。融解は37℃で行い、
ガラス化液の除去は20％ウシ胎仔血清加199培養液中で
行った。なお、何ら凍結処理を施さない胚を対照区とし
た。3. Vitrification preservation embryos were equilibrated in 2% ethylene glycol solution for 12-16 minutes, immersed in the vitrification solution for 60 seconds, and dropped into liquid nitrogen.
Some embryos were filled in a plastic straw of 0.25 cc and then injected into liquid nitrogen. Thawing is performed at 37 ° C.
The removal of the vitrification liquid was performed in a 199 culture medium supplemented with 20% fetal bovine serum. Embryos not subjected to any freezing treatment were used as control groups.
【００２１】4．生存性の判定 融解後の胚を６日間培養し、その胚盤胞期及び孵化胚盤
胞期への発生率を、生存性を判定する指標とした。4. Determination of viability The thawed embryos were cultured for 6 days, and the incidence at the blastocyst stage and hatching blastocyst stage was used as an index for determining viability.
【００２２】5．結果 胚盤胞期及び孵化胚盤胞期への発生率は、本発明のマイ
クロドロップレット法の場合は胚盤胞期への発生率が7
1.2％（37／52）、孵化胚盤胞期への発生率が50.0％（2
6／52）であり、ストロー法の場合は胚盤胞期への発生
率が43.9％（18／41）、孵化胚盤胞期への発生率が17.1
％（7／41）であり、対照区の胚の場合は胚盤胞期への
発生率が74.3％（29／39）、孵化胚盤胞期への発生率が
51.3％（20／39）であった。従って、本発明の方法（マ
イクロドロップレット法）は従来のストロー法に比べて
有意に発生率が高く、牛体外受精３日目胚に対し、対照
区の胚と比較しても遜色のないレベルの生存性を示した
（表２）。5. Results The incidence rates at the blastocyst stage and the hatched blastocyst stage were 7% for the microdroplet method of the present invention.
1.2% (37/52), 50.0% (2
In the case of the straw method, the incidence in the blastocyst stage was 43.9% (18/41), and the incidence in the hatching blastocyst stage was 17.1
% (7/41), and the incidence in the blastocyst stage was 74.3% (29/39) in the case of the control embryo, and the incidence in the hatching blastocyst stage.
51.3% (20/39). Therefore, the method of the present invention (microdroplet method) has a significantly higher incidence than the conventional straw method, and has a level comparable to that of the control group embryo compared to the embryo of the third day of bovine in vitro fertilization. (Table 2).
【００２３】[0023]
【表２】 [Table 2]
【００２４】[0024]
【発明の効果】本発明により、細胞の凍結保存方法が提
供される。本発明の方法によれば、これまでのガラス化
法手間保存が困難であった細胞の凍結保存が可能であ
り、融解後の生存率も高く、極めて有用である。According to the present invention, a method for cryopreserving cells is provided. According to the method of the present invention, it is possible to cryopreserve cells, which has been difficult to save by the vitrification method, and the viability after thawing is high, which is extremely useful.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 志水 学 茨城県つくば市吾妻１−16−２ 401− 521 (72)発明者 クルジストフ アントニ パピス ポーランド国 05−551 ムロコウ，ヤ シュトルツェビエック，ユーエル．ラコ ヴァ １／２ (56)参考文献 特開 昭63−74480（ＪＰ，Ａ) (58)調査した分野(Int.Cl.7，ＤＢ名) C12N 1/00 - 7/08 A01N 1/00 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Manabu Shimizu 1-16-2, Azuma, Tsukuba, Ibaraki Prefecture 401-521 (72) Inventor Kurdishtov Antoni Papis Poland 05-551 Murokov, Ya Stolzebiek, Euel. Lakova 1/2 (56) References JP-A-63-74480 (JP, A) (58) Fields investigated (Int. Cl. 7 , DB name) C12N 1/00-7/08 A01N 1/00
Claims (2)
中に滴下することを特徴とする細胞の凍結保存方法。1. A method for cryopreserving cells, wherein the cells are dropped into liquid nitrogen in the presence of a vitrification solution.
細胞である請求項1記載の保存方法。2. The method according to claim 1, wherein the cells are germ cells or somatic cells of animals or humans.
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