JP4977389B2 - Cell preservation solution and cell preservation method - Google Patents
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本発明は、細胞を保存するために用いる細胞保存液及び細胞保存方法に関する。 The present invention relates to a cell preservation solution and a cell preservation method used for preserving cells.
細胞の長期保存は、一般的には、細胞に液状の保存用媒体を添加し凍結保存させることにより行われる。液状の保存用媒体としては、特許文献１記載の細胞保存液が知られている。この細胞保存液は、細胞中の核酸を劣化させずに細胞を長期間冷凍保存し、解凍後に細胞を培養するためのものであり、５体積％以上２０体積％以下のジメチルスルホキシドを含有する。
理化学研究所バイオリソースセンターや微生物寄託センター等の細胞バンクから分譲された細胞を特許文献１記載の細胞保存液で保存することにより、分譲を受けた者は用時まで細胞を安定に保存することができる。しかしながら、細胞の分譲を受けた者がさらに他の者（第三者）にこの細胞を提供する場合は、第三者がこの細胞を実質的に培養できないような状態で提供することが求められる。第三者に提供された細胞が培養可能な状態であると、細胞バンクの関知しないところで第三者が細胞を培養し、不特定多数の人にこの細胞を分譲する危険性があるからである。
特許文献１の細胞保存液は上述したように解凍後の再培養を目的としたものであるため、第三者に提供する際の細胞保存用媒体としては好ましくない。従って、解凍後の細胞培養が困難であり、且つ細胞を長期間保存することのできる細胞保存液及び細胞保存方法の開発が望まれている。
本発明はこのような事情に鑑みてなされたものであり、細胞を長期間冷凍保存することができ、且つ解凍後に再培養することが困難である細胞保存液及びこれを用いた細胞保存方法を提供することを目的としている。
By storing cells distributed from cell banks such as RIKEN BioResource Center and Microorganism Depositary Center with the cell preservation solution described in Patent Document 1, a person who has received the distribution can stably store the cells until use. it can. However, if the person who received the cell distribution provides this cell to another person (a third party), it is required that the cell is provided in such a state that the third party cannot substantially culture the cell. . If the cell provided to a third party is in a state where it can be cultured, there is a risk that the third party will cultivate the cell without knowing the cell bank and distribute this cell to an unspecified number of people. .
Since the cell preservation solution of Patent Document 1 is intended for re-culture after thawing as described above, it is not preferable as a cell preservation medium when provided to a third party. Accordingly, it is desired to develop a cell preservation solution and a cell preservation method that are difficult to culture after thawing and that can preserve cells for a long period of time.
The present invention has been made in view of such circumstances, and provides a cell preservation solution that can be stored frozen for a long period of time and is difficult to re-culture after thawing, and a cell preservation method using the same. It is intended to provide.
本発明は上記目的を達成するために次の技術的手段を講じた。
本発明の細胞保存液は、細胞を保存するための保存液であって、
ジメチルスルホキシドとｐＨを調整するための緩衝剤とを含有し、ｐＨが３以上４以下である細胞保存液であることを特徴としている。
この細胞保存液によれば、ジメチルスルホキシドの作用により、凍結や融解の際に細胞膜が破壊されにくく、細胞の形態を保ったままの状態で細胞を長期間冷凍保存することが可能となる。また、ｐＨが３以上４以下であるので、核酸の保存安定性を高めることができ、また細胞を解凍した後に培養しても増殖しにくい状態にすることができる。
前記細胞保存液中のジメチルスルホキシドの濃度が１０〜３０体積％であることが好ましい。
前記緩衝剤が、グリシン−塩酸緩衝剤であることが好ましい。
In order to achieve the above object, the present invention takes the following technical means.
The cell preservation solution of the present invention is a preservation solution for preserving cells,
It is a cell preservation solution containing dimethyl sulfoxide and a buffer for adjusting pH, and having a pH of 3 or more and 4 or less.
According to this cell preservation solution, due to the action of dimethyl sulfoxide, the cell membrane is hardly broken during freezing and thawing, and the cells can be stored frozen for a long period of time while maintaining the cell morphology. Further, since the pH is 3 or more and 4 or less, the storage stability of the nucleic acid can be improved, and even if the cells are thawed and cultured, they can be made difficult to grow .
It is preferable that the concentration of dimethyl sulfoxide in the cell preservation solution is 10 to 30% by volume.
The buffer is preferably a glycine-hydrochloric acid buffer.
また、本発明の細胞保存方法は、ｐＨが３以上４以下であり、且つジメチルスルホキシドとｐＨを調整するための緩衝剤とが存在する条件下で細胞を冷凍保存する細胞保存方法であることを特徴としている。
ジメチルスルホキシドの存在下で細胞を保存することにより、凍結や融解の際に細胞膜が破壊されにくく、細胞の形態を保ったままの状態で細胞を長期間冷凍保存することが可能となる。また、ｐＨを３以上４以下とすることで、細胞に含まれる核酸を安定的に保存することができる。また、この細胞保存液を用いて細胞を冷凍保存すると、解凍した後に培養しても増殖しにくい状態にすることができる。
The cell storage method of the present invention is a cell storage method in which cells are frozen and stored under conditions where the pH is 3 or more and 4 or less and dimethyl sulfoxide and a buffer for adjusting pH are present. It is a feature.
By storing the cells in the presence of dimethyl sulfoxide, the cell membrane is hardly broken during freezing and thawing, and the cells can be stored frozen for a long period of time while maintaining the cell morphology. In addition, by setting 3 to 4 the pH, it is possible to store the nucleic acids contained in cells stably. Moreover, if cells are stored frozen using this cell preservation solution, they can be made difficult to proliferate even if they are cultured after thawing.
本発明によれば、細胞を長期間冷凍保存することができ、且つ解凍後に再培養することが困難である細胞保存液及びこれを用いた細胞保存方法を得ることができる。 ADVANTAGE OF THE INVENTION According to this invention, the cell preservation solution which can preserve | save a cell frozen for a long period of time, and is difficult to re-culture after thawing | decompression, and the cell preservation | save method using the same can be obtained.
本発明の細胞保存液は、ジメチルスルホキシドを含有し、ｐＨは３以上４以下に調整されている。ジメチルスルホキシドの作用により、凍結や融解の際に細胞膜が破壊されにくく、細胞の形態を保ったままの状態で細胞を長期間冷凍保存することが可能となる。
ＤＭＳＯは、動物や陸上植物の細胞をはじめ、さまざまな細胞の凍結保存に使われていることから、好ましく用いられる。ＤＭＳＯとしては、一般的に知られている市販のものを用いることができる。細胞保存液中のＤＭＳＯの濃度は、通常１〜５０体積％であり、好ましくは１０〜３０体積％である。濃度が１体積％未満では凍害防御の作用が弱く、また反対に５０体積％を超えても効果は変わらないからである。
細胞保存液は酸性の液体である。細胞保存液のｐＨは３〜４である。ｐＨをこの範囲にすることにより、細胞に含まれる核酸（ＤＮＡ及びＲＮＡ）を安定的に保存することができる。
またこの細胞保存液を用いて細胞を凍結保存すると、解凍した後に保存後の細胞を培養しても増殖しにくい状態にすることができる。解凍後の増殖が起こりにくい理由は不明であるが、細胞が酸性液中で保存されることにより、細胞の増殖に関連するタンパク質が失活する等の要因が考えられる。
細胞保存液の溶媒は特に限定されず、ＤＭＳＯが溶解するものであれば何れも用いることができ、精製水、ＴＥバッファーなどが例示される。
The cell preservation solution of the present invention contains dimethyl sulfoxide, and the pH is adjusted to 3 or more and 4 or less. The action of dimethyl sulfoxide makes it difficult for the cell membrane to be destroyed during freezing and thawing, and the cells can be stored frozen for a long period of time while maintaining the cell morphology.
DMSO is preferably used because it is used for cryopreservation of various cells including cells of animals and land plants. As DMSO, generally known commercially available products can be used. The concentration of DMSO in the cell preservation solution is usually 1 to 50% by volume, preferably 10 to 30% by volume. This is because if the concentration is less than 1% by volume, the effect of protection against freezing damage is weak, and conversely if it exceeds 50% by volume, the effect does not change.
The cell preservation solution is an acidic liquid. The pH of the cell preservation solution is 3-4. By setting the pH within this range, nucleic acids (DNA and RNA) contained in cells can be stably stored.
In addition, if cells are stored frozen using this cell preservation solution, they can be made difficult to proliferate even if the preserved cells are cultured after thawing. The reason why proliferation after thawing is difficult to occur is unknown, but factors such as inactivation of proteins associated with cell proliferation are conceivable when cells are stored in an acidic solution.
The solvent for the cell preservation solution is not particularly limited, and any solvent that can dissolve DMSO can be used, and examples thereof include purified water and TE buffer.
細胞保存液は、ｐＨを調整するための緩衝剤を含有することが好ましい。緩衝剤は、ｐＨを２．５以上６未満に調整できるものであればその種類は特に限定されない。緩衝剤として、例えば、グリシン−塩酸緩衝剤、グッド緩衝剤、リン酸カルシウムなどが挙げられ、好ましくはグリシン−塩酸緩衝剤である。 The cell preservation solution preferably contains a buffer for adjusting the pH. The type of the buffer is not particularly limited as long as the pH can be adjusted to 2.5 or more and less than 6. Examples of the buffer include glycine-hydrochloric acid buffer, Good buffer, calcium phosphate, and the like, and preferably glycine-hydrochloric acid buffer.
この細胞保存液に細胞を添加し凍結させると、半永久的に細胞を保存することができる。凍結の際は、徐々に凝固点まで温度を下げてもよいし、液体窒素等を用いて急速に凍結してもよい。また、融解の際は室温下で静置するなど、徐々に融解してもよいし、加熱するなどして急速に融解してもよい。 When cells are added to this cell preservation solution and frozen, the cells can be preserved semipermanently. During freezing, the temperature may be gradually lowered to the freezing point, or may be rapidly frozen using liquid nitrogen or the like. Further, when melting, it may be gradually melted, for example, by standing at room temperature, or rapidly melted by heating.
保存対象となる細胞の種類は特に限定されない。本発明の細胞保存液を用いると、例えば真核細胞や原核細胞などの長期保存を行うことができる。また、保存する細胞は細胞保存液中に分散されていることが好ましいが、多細胞生物から採取した組織などの細胞塊をそのまま保存することも可能である。 The type of cell to be stored is not particularly limited. When the cell preservation solution of the present invention is used, for example, eukaryotic cells and prokaryotic cells can be preserved for a long time. Further, the cells to be preserved are preferably dispersed in a cell preservation solution, but it is also possible to preserve a cell mass such as a tissue collected from a multicellular organism as it is.
細胞を保存する際の温度としては、細胞保存液が凍結する温度であれば特に限定されないが、−４℃以下であることが好ましく、−２０℃以下であることがより好ましい。 The temperature at which the cells are stored is not particularly limited as long as the cell preservation solution is frozen, but is preferably −4 ° C. or lower, more preferably −20 ° C. or lower.
さらに、本発明の細胞保存方法は、ｐＨが３以上４以下であり、且つジメチルスルホキシドと、ｐＨを調整するための緩衝剤とが存在する条件下で細胞を冷凍保存する細胞保存方法である。この細胞保存方法によれば、ジメチルスルホキシドの作用により、凍結や融解の際に細胞膜が破壊されにくく、細胞の形態を保ったままの状態で細胞を長期間冷凍保存することが可能となる。また、ｐＨ３以上４以下の条件下で保存することにより、細胞中の核酸を安定的に保存することができる。またこの細胞保存方法により保存された細胞は、解凍した後に培養しても増殖しにくい状態となっている。
Furthermore, the cell storage method of the present invention is a cell storage method in which cells are stored frozen under conditions where the pH is 3 or more and 4 or less and dimethyl sulfoxide and a buffer for adjusting the pH are present. According to this cell storage method, due to the action of dimethyl sulfoxide, the cell membrane is not easily destroyed during freezing and thawing, and the cells can be stored frozen for a long period of time while maintaining the cell morphology. Further, by storing under conditions of pH 3 to 4, it is possible to store the nucleic acids in a cell stably. Further, the cells preserved by this cell preservation method are in a state where they are difficult to proliferate even if they are cultured after thawing.
以下の実施例により、本発明をさらに詳細に説明する。
（実施例１）
細胞保存液の細胞保存安定性におけるｐＨ依存性を検討した。
細胞保存液として、ｐＨを２、３、３．５、４、６及び８に調整した６種類の細胞保存液を用いた。ｐＨが２、３、３．５及び４である細胞保存液は、２０％ ＤＭＳＯ及びグリシン（２００ｍＭ）−塩酸を含み、塩酸の量でｐＨを調整した。ｐＨが６及び８である細胞保存液は、２０％ ＤＭＳＯ及びＴｒｉｓ（２００ｍＭ）−塩酸を含み、塩酸の量でｐＨを調整した。
ヒト肺癌由来の培養細胞（ＬＣ−２／ａｄ細胞）（５×１０５ｃｅｌｌｓ）を各ｐＨの細胞保存液（−２０℃）で１日冷凍保存した。解凍した後、遠心して細胞を沈降させ、細胞保存液を除去した。ここに２００μｌのホモジナイズ試薬（シスメックス（株）製）を加えて破砕し、１０倍希釈して測定用試料を調製した。ＧＤ−１００（シスメックス（株）製）を用いてこの測定用試料とサイトケラチン試薬（シスメックス（株）製）とを混合し、試料中のサイトケラチン１９（ＣＫ１９）ｍＲＮＡを定量した。定量結果を図１に示す。
図１より、ｐＨ３以上６未満の細胞保存液で細胞を保存した場合に、多くのコピー数のｍＲＮＡが検出された。即ち、ｐＨ３以上６未満の細胞保存液中では、細胞のＲＮＡの安定性が良好であることがわかった。
得られた図１のグラフのデータから、細胞保存液のｐＨと細胞に含まれるＲＮＡの保存安定性には相関関係があることが確認された。これによると、ｐＨ２．５〜５．５の細胞保存液を用いると細胞中のＲＮＡを安定的に保存でき、ｐＨ３〜４の細胞保存液を用いると最も安定的にＲＮＡを保存できることがわかった。
The following examples illustrate the invention in more detail.
Example 1
The pH dependence of cell preservation stability of the cell preservation solution was examined.
As the cell preservation solution, six kinds of cell preservation solutions adjusted to pH 2, 3, 3.5, 4, 6 and 8 were used. Cell preservation solutions having pHs of 2, 3, 3.5 and 4 contained 20% DMSO and glycine (200 mM) -hydrochloric acid, and the pH was adjusted with the amount of hydrochloric acid. The cell preservation solution having a pH of 6 and 8 contained 20% DMSO and Tris (200 mM) -hydrochloric acid, and the pH was adjusted with the amount of hydrochloric acid.
Cultured cells derived from human lung cancer (LC-2 / ad cells) (5 × 10 5 cells) were frozen and stored in a cell storage solution (−20 ° C.) at each pH for 1 day. After thawing, the cells were sedimented by centrifugation, and the cell preservation solution was removed. 200 μl of a homogenizing reagent (manufactured by Sysmex Co., Ltd.) was added and crushed, and diluted 10 times to prepare a measurement sample. The sample for measurement and cytokeratin reagent (manufactured by Sysmex Corporation) were mixed using GD-100 (manufactured by Sysmex Corporation), and cytokeratin 19 (CK19) mRNA in the sample was quantified. The quantitative results are shown in FIG.
As shown in FIG. 1, when the cells were stored in a cell storage solution having a pH of 3 or more and less than 6, a large number of copies of mRNA was detected. That is, it was found that the stability of cellular RNA was good in a cell preservation solution having a pH of 3 or more and less than 6.
From the data of the graph of FIG. 1 obtained, it was confirmed that there was a correlation between the pH of the cell preservation solution and the storage stability of RNA contained in the cells. According to this, it was found that RNA in cells can be stably stored using a cell preservation solution of pH 2.5 to 5.5, and RNA can be stored most stably using a cell preservation solution of pH 3 to 4. .
（実施例２）
次に、ｐＨ３．５の細胞保存液を用いて細胞保存安定性を検討した。この細胞保存液は、上述のように２０％ ＤＭＳＯ及びグリシン（２００ｍＭ）−塩酸を含み、塩酸の量でｐＨを３．５に調整したものである。
ＬＣ−２／ａｄ細胞（０．８×１０５ｃｅｌｌｓ／ｔｕｂｅ）を、１ｍｌの細胞保存液（ｐＨ３．５、−２０℃）に懸濁し、凍結保存した。これを０、１５、４８、５６、８３又は１０５日間保存した後、１２００ｒｐｍで遠心して細胞保存液を除去した。ここに２００μｌのホモジナイズ試薬（シスメックス（株）製）を加え、２２Ｇシリンジ、３０ストローク、氷上で細胞を破砕し、−８０℃で一時保存した。その後、この細胞破砕液を２００倍希釈（４×１０３ｃｅｌｌｓ／ｔｅｓｔ）して測定用試料を調製し、実施例１と同様にしてＣＫ１９ｍＲＮＡを定量した。同様の実験を３回繰り返し（ｎ＝３）、その平均値を図２のグラフ（◆）に示した。
ポジティブコントロールとして、ＬＣ−２／ａｄ細胞（０．８×１０５ｃｅｌｌｓ／ｔｕｂｅ）を−８０℃で凍結細胞ペレットに保存したものを用い、実施例１と同様に処理してＣＫ１９ｍＲＮＡ量を測定した。同様の実験を２回繰り返し（ｎ＝２）、その平均値を図２のグラフ（■）に示した。
図２より、本発明の細胞保存液を用いて細胞を保存した場合、少なくとも−２０℃で１０５日間保存しても、−８０℃で保存した細胞（ポジティブコントロール）と同程度にＲＮＡを保存できることがわかった。
(Example 2)
Next, cell storage stability was examined using a cell storage solution having a pH of 3.5. This cell preservation solution contains 20% DMSO and glycine (200 mM) -hydrochloric acid as described above, and the pH is adjusted to 3.5 with the amount of hydrochloric acid.
LC-2 / ad cells (0.8 × 10 5 cells / tube) were suspended in 1 ml of cell preservation solution (pH 3.5, −20 ° C.) and stored frozen. This was stored for 0, 15, 48, 56, 83, or 105 days, and then centrifuged at 1200 rpm to remove the cell storage solution. 200 μl of a homogenizing reagent (manufactured by Sysmex Corporation) was added thereto, and the cells were crushed on a 22 G syringe, 30 strokes on ice, and temporarily stored at −80 ° C. Thereafter, this cell disruption solution was diluted 200-fold (4 × 10 3 cells / test) to prepare a measurement sample, and CK19 mRNA was quantified in the same manner as in Example 1. The same experiment was repeated three times (n = 3), and the average value is shown in the graph (♦) in FIG.
As a positive control, LC-2 / ad cells (0.8 × 10 5 cells / tube) stored in a frozen cell pellet at −80 ° C. were treated in the same manner as in Example 1 to measure the amount of CK19 mRNA. . The same experiment was repeated twice (n = 2), and the average value is shown in the graph (■) in FIG.
FIG. 2 shows that when cells are stored using the cell preservation solution of the present invention, RNA can be stored to the same extent as cells stored at −80 ° C. (positive control) even if stored at −20 ° C. for 105 days. I understood.
（実施例３）
次に、本発明の細胞保存液で凍結保存した細胞が解凍後培養せず、細胞形態を保持しているかどうかを確認した。
ＬＣ−２／ａｄ細胞（１×１０５ｃｅｌｌｓ）を、１）実施例２と同じ細胞保存液（ｐＨ３．５）、２）十慈フィールド（株）製セルバンカー（商品名）、３）培地（（ＨａｍＦ１２：ＲＰＭＩ１６４０＝１：１）、１０％ ＦＢＳ、及び２５ｍＭ ＨＥＰＥＳを含む）で５日間保存した（いずれも−２０℃）。全量を７５ｃｍ２のフラスコにて培地（（ＨａｍＦ１２：ＲＰＭＩ１６４０＝１：１）、１０％ ＦＢＳ、及び２５ｍＭ ＨＥＰＥＳを含む）で培養した。培養開始１日後及び４日後に顕微鏡で観察して写真撮影を行った。その後、培地を交換し、１３日目に再度顕微鏡で観察して写真撮影を行った。図３にそれぞれの培養開始後１日目、４日目、及び１３日目の顕微鏡観察像を示し、図４に培地及び細胞保存液中で保存した細胞の１３日目の拡大画像を示す。
図３から、セルバンカーで保存した細胞は、解凍後の培養により、１３日目には細胞がプレートに接着して増殖していることが確認されたが、培地及び細胞保存液で保存した細胞は、１３日目にも増殖していなかった。
また、図４から、培地で保存した細胞は、細胞膜が壊れたものが多かったが、細胞保存液で保存した細胞は、細胞膜が損傷せずに細胞の形態が保たれた状態であった。
以上より、本実施例の細胞保存液で細胞を凍結保存すると、解凍した後に実質的に再培養できず、且つ細胞の形態を保ったまま保存されることが確認された。
Example 3
Next, it was confirmed whether the cells cryopreserved with the cell preservation solution of the present invention were not cultured after thawing and retained the cell morphology.
LC-2 / ad cells (1 × 10 5 cells) 1) Cell storage solution (pH 3.5) same as Example 2 2) Cell banker (trade name) manufactured by Toji Field Co., Ltd. 3) Medium (HamF12: RPMI1640 = 1: 1), containing 10% FBS, and 25 mM HEPES) for 5 days (both at −20 ° C.). The whole amount was cultured in a medium ((HamF12: RPMI1640 = 1: 1), containing 10% FBS and 25 mM HEPES) in a 75 cm 2 flask. After 1 day and 4 days from the start of the culture, photographs were taken under a microscope. Thereafter, the medium was changed, and on the 13th day, the photograph was taken again by observation with a microscope. FIG. 3 shows microscopic images of the first, fourth and thirteenth days after the start of each culture, and FIG. 4 shows enlarged images of the thirteenth day of cells stored in the medium and the cell preservation solution.
From FIG. 3, it was confirmed that the cells stored in the cell banker were grown on the 13th day by culturing after thawing. However, the cells stored in the medium and the cell storage solution were confirmed. Was not growing on day 13.
Further, from FIG. 4, many cells stored in the culture medium had broken cell membranes, but the cells stored in the cell storage solution were in a state where the cell morphology was maintained without damaging the cell membrane.
From the above, it was confirmed that when cells were cryopreserved with the cell preservation solution of this example, they could not be substantially recultured after thawing and were preserved while maintaining the cell morphology.
（実施例４）
細胞保存液で凍結保存した細胞を解凍し、培養するか否かを確認した。
ＬＣ−２／ａｄ細胞を、実施例２と同じ細胞保存液（ｐＨ３．５、−２０℃）に５日間−２０℃で冷凍保存した。解凍後、遠心して細胞保存液を除去し、ＬＣ−２／ａｄ細胞を回収した。この細胞を計測し、１×１０４ｃｅｌｌｓ／ｗｅｌｌ（９６ウェルプレート）及び５×１０４ｃｅｌｌｓ／ｗｅｌｌ（２４ウェルプレート）となるよう各ウェルに解凍後の細胞を収容し、培養を開始した。１〜１３日間培養し、各ウェル内の細胞を回収して細胞計数板にて計数した（９６ウェルプレートに関してはｎ＝３、２４ウェルプレートに関してはｎ＝２で計数した）。
対照として、冷凍保存していないＬＣ−２／ａｄ細胞を用い、上記と同様に培養した。但し、ここでは培養は１〜８日間だけ行った。これらの結果を図５及び図６に示す。図５及び図６において、■は本実施例の細胞保存液を用いた際の実験結果を示し、◆は対照実験結果である。図５は、９６ウェルプレートにおける結果であり、図６は２４ウェルプレートにおける結果である。
図５及び図６より、冷凍保存していない細胞は培養により増殖したが、本実施例の細胞保存液で凍結保存した細胞は、解凍後に培養しても、１３日間ほとんど増殖しないことがわかった。
Example 4
It was confirmed whether the cell cryopreserved with the cell preservation solution was thawed and cultured.
LC-2 / ad cells were stored frozen at −20 ° C. for 5 days in the same cell preservation solution (pH 3.5, −20 ° C.) as in Example 2. After thawing, the cell preservation solution was removed by centrifugation, and LC-2 / ad cells were collected. The cells were counted, and the thawed cells were accommodated in each well so as to be 1 × 10 4 cells / well (96-well plate) and 5 × 10 4 cells / well (24-well plate), and culture was started. The cells were cultured for 1 to 13 days, and the cells in each well were collected and counted on a cell counter (n = 3 for a 96-well plate and n = 2 for a 24-well plate).
As a control, LC-2 / ad cells that had not been cryopreserved were used and cultured as described above. However, here, culture was performed only for 1 to 8 days. These results are shown in FIGS. In FIGS. 5 and 6, ▪ represents experimental results when using the cell preservation solution of this example, and ◆ represents control experimental results. FIG. 5 shows the results in a 96-well plate, and FIG. 6 shows the results in a 24-well plate.
From FIG. 5 and FIG. 6, it was found that cells that had not been cryopreserved proliferated by culturing, but cells that had been cryopreserved with the cell preservation solution of this Example hardly proliferated for 13 days even when cultured after thawing. .
（実施例５）
さらに、細胞保存液を用いて５日間−２０℃で凍結保存したＬＣ−２／ａｄ細胞を解凍後にＤＮＡ染色色素であるヘキスト３３３４２により染色した。また、培養フラスコで培養したＬＣ−２／ａｄ細胞を、凍結保存せずにトリプシン処理を行い、フラスコから剥離し、これをポジティブコントロールとして用いた。これらの位相差像及び蛍光像を図７に示す。
図７から、ポジティブコントロールと同様に細胞保存液で保存した細胞もＤＮＡが染色されていることから、本発明の細胞保存液を用いると、ＤＮＡを劣化させずに細胞を保存できることがわかった。
(Example 5)
Furthermore, LC-2 / ad cells cryopreserved at −20 ° C. for 5 days using a cell preservation solution were thawed and then stained with Hoechst 33342, which is a DNA staining dye. In addition, LC-2 / ad cells cultured in a culture flask were treated with trypsin without being cryopreserved, detached from the flask, and used as a positive control. These phase difference images and fluorescence images are shown in FIG.
From FIG. 7, it was found that cells stored in the cell preservation solution as well as the positive control were stained with DNA, and therefore, using the cell preservation solution of the present invention, it was possible to preserve the cells without degrading the DNA.
Claims (4)
ジメチルスルホキシドとｐＨを調整するための緩衝剤とを含有し、ｐＨが３以上４以下である細胞保存液。 A storage solution for storing cells,
A cell preservation solution containing dimethyl sulfoxide and a buffer for adjusting pH and having a pH of 3 or more and 4 or less.
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