JPH07255469A - Frozen storage solution for mammalian cell - Google Patents

Frozen storage solution for mammalian cell

Info

Publication number
JPH07255469A
JPH07255469A JP6046859A JP4685994A JPH07255469A JP H07255469 A JPH07255469 A JP H07255469A JP 6046859 A JP6046859 A JP 6046859A JP 4685994 A JP4685994 A JP 4685994A JP H07255469 A JPH07255469 A JP H07255469A
Authority
JP
Japan
Prior art keywords
cells
weight
cryopreservation
added
keratin
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.)
Pending
Application number
JP6046859A
Other languages
Japanese (ja)
Inventor
Shigeaki Hirao
滋章 平尾
Ryohei Yamamoto
良平 山本
Toyokazu Nishino
豊和 西野
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kurabo Industries Ltd
Kurashiki Spinning Co Ltd
Original Assignee
Kurabo Industries Ltd
Kurashiki Spinning Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Kurabo Industries Ltd, Kurashiki Spinning Co Ltd filed Critical Kurabo Industries Ltd
Priority to JP6046859A priority Critical patent/JPH07255469A/en
Publication of JPH07255469A publication Critical patent/JPH07255469A/en
Pending legal-status Critical Current

Links

Abstract

PURPOSE:To prepare a solution for refrigerating mammalian cells, which contains a specific protein such as keratin hydrolyzate and is useful for refrigeration of hepatic cells or the like because it gives good survival rate during the refrigeration. CONSTITUTION:Dimethyl sulfoxide(DMSO) is added to the base medium for the cell culture, further keratin hydrolyzate, gelatin hydrolyzate or serum albumen is added. The hydrolyzed keratin or the gelatin hydrolyzate is added in an amount of more than 0.5wt.%, preferably 5 to 15wt.%, while serum albumen is added in an amount of more than 0.5wt.%, preferably more than 2.5wt.%. The survival rate of the cells after freezing and thawing is more improved than in using a storage solution containing only DMSO or DMSO and serum.

Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【従来の技術】動物細胞を凍結状態で長期間保存する技
術は細胞の研究や有効利用のためには不可欠である。し
かしながら、凍結、融解に伴う浸透圧の変化等により、
細胞が障害され、融解後の生存率が悪化したり機能が維
持できなかったりする。このような凍結障害を防止する
ための凍結障害防護剤として、従来からグリセリン、ジ
メチルスルホキシドおよび血清を含む培地が広く使用さ
れている。これらの保護剤を用いても融解後に得られる
生存率は満足できるものではなく、特に肝細胞のごとき
比較的大きな細胞を凍結保存する場合には凍結−融解後
の細胞の生存率はかなり低くなる。
2. Description of the Related Art A technique for storing animal cells in a frozen state for a long period of time is indispensable for cell research and effective use. However, due to changes in osmotic pressure due to freezing and thawing,
The cells are damaged, and the survival rate after thawing is deteriorated or the function cannot be maintained. Conventionally, a medium containing glycerin, dimethylsulfoxide and serum has been widely used as a cryoprotective agent for preventing such freeze damage. Even if these protective agents are used, the viability obtained after thawing is not satisfactory, and especially when relatively large cells such as hepatocytes are cryopreserved, the viability of the cells after freeze-thawing becomes considerably low. .

【0002】生存率を上昇させるため、血清に代えてメ
チルセルロースあるいはゼラチンを添加して凍結保護剤
とする方法が提案されている(特開昭63−21647
6、特開平5−7489)。しかしながら、これらの添
加剤は高濃度となると粘性が高くなったりゲル化したり
するため、使用できる濃度は限定されるが、当該濃度で
は特に肝細胞のごとき大型の細胞の凍結保存には不十分
である。また、血清濃度を50重量%程度まで上げるこ
とにより凍結障害を減少させる方法も提案されている
が、この方法によっても肝細胞等の凍結保存において
は、融解後の生存率が低い。
In order to increase the survival rate, a method has been proposed in which methylcellulose or gelatin is added in place of serum to provide a cryoprotective agent (Japanese Patent Laid-Open No. 63-21647).
6, JP-A-5-7489). However, since these additives have a high viscosity and gelate at a high concentration, the usable concentration is limited, but the concentration is not sufficient for cryopreservation of large cells such as hepatocytes. is there. Further, a method of reducing freezing damage by increasing the serum concentration to about 50% by weight has been proposed, but this method also has a low survival rate after thawing in cryopreservation of hepatocytes and the like.

【0003】[0003]

【発明が解決しようとする課題】すなわち本発明は、動
物細胞の凍結保存において良好な生存率を与える凍結保
存液を提供することを目的とする。
That is, an object of the present invention is to provide a cryopreservation liquid which gives a good survival rate in cryopreservation of animal cells.

【0004】[0004]

【課題を解決するための手段】本発明はケラチン加水分
解物、加水分解ゼラチンおよび血清アルブミンからなる
群から選択されるタンパク質を0.5重量%以上含有す
る動物細胞の凍結保存液に関する。
The present invention relates to a cryopreservation solution for animal cells containing 0.5% by weight or more of a protein selected from the group consisting of keratin hydrolyzate, hydrolyzed gelatin and serum albumin.

【0005】本発明の凍結保存液に用いるケラチン加水
分解物としては、可溶化ウールケラチンが好ましい。可
溶化ウールケラチンは羊毛を加水分解して得られるもの
であり、典型的には30%過酸化水素液で1時間酸化分
解し、得られた可溶化ウールケラチン溶液を酢酸でpH
5に調整し、これに2倍量のアルコールを添加し、生じ
た沈殿を集めて乾燥し、その後蒸留水に溶解して不純物
を除いたのち再び乾燥して得られるものを用ればよい。
本発明に用いるケラチン加水分解物の平均分子量は1〜
2万である。またそのアミノ酸組成は、システインが消
失する以外は原料の羊毛ケラチンと同じである。
As the keratin hydrolyzate used in the cryopreservation solution of the present invention, solubilized wool keratin is preferable. Solubilized wool keratin is obtained by hydrolyzing wool, and is typically oxidatively decomposed with a 30% hydrogen peroxide solution for 1 hour, and the obtained solubilized wool keratin solution is treated with acetic acid to pH.
The amount obtained is adjusted to 5, and a double amount of alcohol is added thereto, and the resulting precipitate is collected and dried, then dissolved in distilled water to remove impurities, and then dried again to be used.
The average molecular weight of the keratin hydrolyzate used in the present invention is 1 to
It is 20,000. The amino acid composition is the same as that of the raw material wool keratin except that cysteine disappears.

【0006】本発明の凍結保存液は加水分解ケラチンを
0.5重量%以上、好ましくは5〜15重量%含有す
る。0.5重量%未満では融解後の生存率が改善され
ず、また15重量%を越えるとかえって生存率が悪くな
る。
The cryopreservation liquid of the present invention contains hydrolyzed keratin in an amount of 0.5% by weight or more, preferably 5 to 15% by weight. If it is less than 0.5% by weight, the survival rate after melting is not improved, and if it exceeds 15% by weight, the survival rate is rather deteriorated.

【0007】本発明の凍結保存液に用いる加水分解ゼラ
チンは、ゼラチンをアルカリ又は酵素により加水分解し
て低分子化したものである。ゼラチンの加水分解をアル
カリにより行う場合は、例えばゼラチンを1M苛性ソー
ダに溶かして10%濃度とし、100℃で1時間加水分
解する。また酵素による場合には10%程度のゼラチン
水溶液を調製し、これをプロテアーゼにて消化する。プ
ロテアーゼとしては例えばアマノ社製のプロテアーゼT
1、プロテアーゼA、パンクレアチンF等が好適に用い
られる。
The hydrolyzed gelatin used in the cryopreservation liquid of the present invention is gelatin hydrolyzed with an alkali or an enzyme to have a low molecular weight. When gelatin is hydrolyzed with an alkali, for example, gelatin is dissolved in 1M caustic soda to a concentration of 10% and hydrolyzed at 100 ° C. for 1 hour. When using an enzyme, a 10% gelatin aqueous solution is prepared and this is digested with protease. Examples of the protease include protease T manufactured by Amano
1 , protease A, pancreatin F and the like are preferably used.

【0008】本発明の加水分解ゼラチンは、分子量30
00〜20000、好ましくは4000〜10000の
ものを用いる。ゼラチンを分解せずに用いたり、分解が
不十分であると、0.5重量%以上濃度では室温でもゲ
ル化し、通常0℃付近で行う細胞の懸濁や分注が不可能
となり、凍結障害保護剤として使用することができな
い。
The hydrolyzed gelatin of the present invention has a molecular weight of 30.
The one used is from 00 to 20000, preferably 4000 to 10000. If gelatin is used without being decomposed or if the decomposition is insufficient, it will gel at a concentration of 0.5% by weight or more even at room temperature, making it impossible to suspend or dispense cells normally performed at around 0 ° C., which causes freezing damage. It cannot be used as a protective agent.

【0009】本発明の凍結保存液中、加水分解ゼラチン
は0.5重量%以上、好ましくは5〜15重量%含有さ
れる。0.5重量%未満では融解後の生存率が改善され
ず、また15重量%を越えるとかえって生存率が悪くな
る。
Hydrolyzed gelatin is contained in the cryopreservation liquid of the present invention in an amount of 0.5% by weight or more, preferably 5 to 15% by weight. If it is less than 0.5% by weight, the survival rate after melting is not improved, and if it exceeds 15% by weight, the survival rate is rather deteriorated.

【0010】本発明の凍結保存液に用いる血清アルブミ
ンは血清中に多く含まれる分子量が5〜6万のタンパク
質である。血清アルブミンの由来は何であっても良い
が、ウシ血清アルブミンが入手しやすく、また経済的で
ある。凍結障害保護剤として血清を添加することは通常
行われているが、血清は様々な成分からなる組成物であ
り、細胞に対する阻害物質も含まれている可能性があ
る。よって、高濃度の血清を添加するよりアルブミンを
単独で添加する方が、かかる阻害物質の影響を受けない
ため好ましい。
Serum albumin used in the cryopreservation solution of the present invention is a protein contained in serum in a large amount and having a molecular weight of 50,000 to 60,000. Serum albumin may be derived from any source, but bovine serum albumin is easily available and economical. Serum is usually added as a cryoprotective agent, but serum is a composition composed of various components and may contain an inhibitory substance for cells. Therefore, it is preferable to add albumin alone rather than adding high-concentration serum because it is not affected by such an inhibitor.

【0011】本発明の凍結保存液には血清アルブミンを
0.5重量%以上、好ましくは2.5重量%以上添加す
る。血清アルブミンの添加量が0.5重量%未満の場合
には生存率の改善がみられず、好ましくない。
Serum albumin is added to the cryopreservation liquid of the present invention in an amount of 0.5% by weight or more, preferably 2.5% by weight or more. If the amount of serum albumin added is less than 0.5% by weight, the survival rate is not improved, which is not preferable.

【0012】本発明の凍結保存液は、細胞培養用の基礎
培地にDMSOを添加したものに上述のタンパク質を含
有する。基礎培地としては、動物細胞培養用に用いられ
ているものであれば特に限定されず、保存する細胞に応
じて選択すればよい。DMSOは終濃度が10%となる
ように添加するのが好ましい。
The cryopreservation liquid of the present invention contains the above-mentioned protein in a basal medium for cell culture to which DMSO is added. The basal medium is not particularly limited as long as it is used for culturing animal cells, and may be selected according to the cells to be stored. DMSO is preferably added so that the final concentration becomes 10%.

【0013】本発明の凍結保存液にはさらにセルロース
誘導体またはポリエチレングリコールから選択される高
分子化合物を含有してもよい。セルロース誘導体として
は例えばメチルセルロース、ヒドロキシメチルセルロー
ス等が好適に用いられる。セルロース誘導体の平均分子
量は〜4000であるものが好適に用いられる。ポリエ
チレングリコールとしては、平均分子量が4000〜2
0000のものが好適に用いられる。本発明の凍結保存
液中、高分子は0.05〜2重量%、好ましくは0.1〜
0.5重量%含有する。高分子の含有量が0.5重量%を
越えると保存液の粘性が高くなり取り扱いが困難とな
る。
The cryopreservation liquid of the present invention may further contain a polymer compound selected from a cellulose derivative or polyethylene glycol. As the cellulose derivative, for example, methyl cellulose, hydroxymethyl cellulose and the like are preferably used. A cellulose derivative having an average molecular weight of 4,000 is preferably used. Polyethylene glycol has an average molecular weight of 4000-2
Those of 0000 are preferably used. In the cryopreservation liquid of the present invention, the polymer content is 0.05 to 2% by weight, preferably 0.1 to 2.
Contains 0.5% by weight. If the content of the polymer exceeds 0.5% by weight, the viscosity of the storage solution becomes high and handling becomes difficult.

【0014】本発明の凍結保存液で保存される細胞は特
に限定されず、培養細胞であっても動物より単離された
細胞であっても好適に保存される。さらに本発明の凍結
保存液を用いれば、肝細胞のごとき比較的大型の細胞で
あっても良好に凍結保存することが可能である。
The cells to be stored in the cryopreservation solution of the present invention are not particularly limited, and they are preferably stored regardless of whether they are cultured cells or cells isolated from animals. Furthermore, by using the cryopreservation liquid of the present invention, it is possible to favorably cryopreserve relatively large cells such as hepatocytes.

【0015】本発明の凍結保存液を用いて細胞を凍結保
存するための具体的な方法は以下の通りである。 1. 所定終濃度の2倍濃度の凍結障害保護剤を含有す
る凍結保存液と、2倍濃度の凍結保存する細胞の懸濁液
を1:1で混合する。凍結保存用細胞懸濁液は、最終的
に細胞濃度が0.5〜10×106個/mlとなるように
調製するのが好ましい。 2. 得られた凍結保存用細胞懸濁液を2ml容の凍結
保存用チューブに1mlずつ分注する。 3. チューブに分注した凍結保存用細胞懸濁液の温度
をおよそ−1℃/分の速度で下げ、凍結する。温度の制
御はプログラムフリーザーを使用することによっておこ
なっても良いが、簡便にはチューブの周囲を綿でくるん
で−70〜−80℃に設定したフリーザーに2時間から
一晩程度放置すれば良い。 4. 最終的にチューブを−80〜−186℃の液体窒
素もしくはフリーザー中で保存する。 5. 細胞を融解する際は、常法に従い37℃の水中に
てすみやかに融解させる。
The specific method for cryopreserving cells using the cryopreservation solution of the present invention is as follows. 1. A cryopreservation solution containing a cryoprotective agent at twice the predetermined final concentration and a suspension of cells to be cryopreserved at twice the concentration are mixed 1: 1. The cell suspension for cryopreservation is preferably prepared so that the final cell concentration will be 0.5 to 10 × 10 6 cells / ml. 2. The obtained cell suspension for cryopreservation is dispensed in 1 ml aliquots into a cryopreservation tube having a volume of 2 ml. 3. The temperature of the cryopreservation cell suspension dispensed in a tube is lowered at a rate of about -1 ° C / min to freeze. The temperature may be controlled by using a program freezer, but it is convenient to wrap the tube with cotton and leave it in the freezer set at -70 to -80 ° C for about 2 hours to overnight. 4. Finally tubes are stored in liquid nitrogen or freezer at -80 to -186 ° C. 5. When thawing the cells, the cells are thawed immediately in water at 37 ° C. according to a conventional method.

【0016】[0016]

【実施例】以下本発明を実施例によりさらに詳細に説明
する。 1.可溶化ウールケラチンの調製 羊毛を30%過酸化水素液で1時間酸化分解し、得られ
た可溶化ウールケラチン溶液を酢酸でpH5に調製し、
これに2倍量のアルコールを添加し、生じた沈殿を集め
て乾燥した。この乾燥物を蒸留水に溶解し、不純物を除
いた後乾燥したものを可溶化ウールケラチンとして用い
た。得られた可溶化ウールケラチンの平均分子量は1〜
2万であった。
EXAMPLES The present invention will now be described in more detail with reference to examples. 1. Preparation of Solubilized Wool Keratin Wool is oxidatively decomposed with 30% hydrogen peroxide solution for 1 hour, and the resulting solubilized wool keratin solution is adjusted to pH 5 with acetic acid,
To this was added twice the amount of alcohol, and the resulting precipitate was collected and dried. The dried product was dissolved in distilled water to remove impurities, and then dried to be used as solubilized wool keratin. The average molecular weight of the obtained solubilized wool keratin is 1 to
It was 20,000.

【0017】2.加水分解ゼラチンの調製 ゼラチン(DIFCO社製)を10〜20%の濃度とな
るよう水に懸濁し、これを60℃に加熱してゼラチンを
溶解させた。ゼラチン溶液を37℃まで冷却し、プロテ
アーゼA(天野社製)を0.1%となるように添加し、
37℃で15〜30分間反応させた。反応後100℃で
20分間加熱し、濾過滅菌、又はオートクレーブ滅菌し
たものを加水分解ゼラチンとして用いた。得られた加水
分解ゼラチンの平均分子量は3000〜20000であ
った。上述の可溶化ウールケラチン、加水分解ゼラチン
および市販のウシ血清アルブミン(オルガノン社製)を
用いて凍結保存液を調製し、ラット肝実質細胞を本発明
の凍結保存液を用いて凍結保存し、融解後の生存率を測
定した
2. Preparation of Hydrolyzed Gelatin Gelatin (manufactured by DIFCO) was suspended in water to a concentration of 10 to 20%, and this was heated to 60 ° C to dissolve the gelatin. The gelatin solution was cooled to 37 ° C., and protease A (manufactured by Amano) was added to 0.1%,
The reaction was carried out at 37 ° C for 15 to 30 minutes. After the reaction, the product was heated at 100 ° C. for 20 minutes, sterilized by filtration or sterilized by autoclave, and used as hydrolyzed gelatin. The average molecular weight of the obtained hydrolyzed gelatin was 3,000 to 20,000. A cryopreservation solution was prepared using the above-mentioned solubilized wool keratin, hydrolyzed gelatin and commercially available bovine serum albumin (manufactured by Organon), and rat liver parenchymal cells were cryopreserved using the cryopreservation solution of the present invention and thawed. After survival rate was measured

【0018】3.ラット肝実質細胞の凍結保存 1)細胞の調製:コラゲナーゼ溶液をラット肝に灌流す
ることにより、肝細胞を分散させ、得られた細胞を遠心
分離して肝実質細胞を精製して肝実質細胞を得た。
3. Cryopreservation of rat liver parenchymal cells 1) Preparation of cells: Hepatocytes were dispersed by perfusing a rat liver with a collagenase solution, and the obtained cells were centrifuged to purify the liver parenchymal cells. Obtained.

【0019】2)保存液の調製:終濃度の2倍の凍結障
害保護剤を含む保存液と終濃度の2倍の細胞を含む細胞
懸濁液を1:1の割合で混合し、細胞の終濃度が6×1
6個/mlの凍結保存用細胞懸濁液を得た。得られた
懸濁液を2ml容の凍結保存用チューブに1mlずつ分
注した。
2) Preparation of stock solution: A stock solution containing a cryoprotective agent at twice the final concentration and a cell suspension containing cells at twice the final concentration were mixed at a ratio of 1: 1 to prepare the cells. Final concentration is 6 × 1
A cell suspension for cryopreservation of 0 6 cells / ml was obtained. The obtained suspension was dispensed in 1 ml portions into 2 ml cryopreservation tubes.

【0020】3)凍結保存:チューブをチューブラック
へ入れ、周囲を綿でくるんだ後、−80℃のフリーザー
内へ一晩置いた。チューブラックを液体窒素中へ移動さ
せ、7日間凍結保存した。
3) Cryopreservation: The tube was placed in a tube rack, wrapped around with cotton, and then placed in a -80 ° C freezer overnight. The tube rack was moved into liquid nitrogen and stored frozen for 7 days.

【0021】4)融解:凍結細胞を液体窒素より取り出
し、37℃の水中でゆるやかに攪拌して融解した。
4) Thaw: Frozen cells were taken out from liquid nitrogen and thawed by gently stirring in water at 37 ° C.

【0022】5)生存率の測定:融解した細胞懸濁液1
mlに対して0.3%トリパンブルーを1mlを添加し
て撹拌し、顕微鏡下、染色細胞と非染色細胞の数をカウ
ントした。生存率は全細胞数に対する非染色細胞数の割
合(%)で示した。
5) Viability measurement: thawed cell suspension 1
1 ml of 0.3% trypan blue was added to ml and stirred, and the number of stained cells and non-stained cells was counted under a microscope. The survival rate was indicated by the ratio (%) of the number of unstained cells to the total number of cells.

【0023】実施例1 基礎培地としてDMSOを10%含有するイスコフ改変
DME培地を用いた。この培地へ、可溶化ウールケラチ
ン、血清アルブミン、加水分解ゼラチン、およびウシ胎
児血清をそれぞれ表1に記載の量含有する凍結保存液を
用いてラットの肝実質細胞を7日間凍結保存後、融解し
た場合の生存率を調べた。結果を表1に示す。
Example 1 An Iscove's modified DME medium containing 10% DMSO was used as a basal medium. Using a cryopreservation solution containing solubilized wool keratin, serum albumin, hydrolyzed gelatin, and fetal bovine serum in the medium in the amounts shown in Table 1, rat hepatocytes were cryopreserved for 7 days and then thawed. The survival rate in each case was investigated. The results are shown in Table 1.

【0024】[0024]

【表1】 [Table 1]

【0025】実施例2 実施例1と同じ基礎培地へDMSO10%と加水分解ゼ
ラチンを0〜20重量%含有する凍結保存液を用いてラ
ットの肝実質細胞を7日間凍結保存後、融解した場合の
生存率を調べた。結果を表2に示す。
Example 2 Rat hepatocytes were cryopreserved for 7 days using a cryopreservation solution containing 10% DMSO and 0-20% by weight of hydrolyzed gelatin in the same basal medium as in Example 1, and then thawed. The survival rate was examined. The results are shown in Table 2.

【0026】[0026]

【表2】 [Table 2]

【0027】実施例3 実施例1と同じ基礎培地へ、DMSO10%、メチルセ
ルロース0.1〜0.2重量%および加水分解ゼラチン0
〜15重量%を含有する凍結保存液を用いてラットの肝
実質細胞を7日間凍結保存後、融解した場合の生存率を
調べた。結果を表3に示す。
Example 3 To the same basal medium as in Example 1, DMSO 10%, methylcellulose 0.1-0.2% by weight and hydrolyzed gelatin 0
Using a cryopreservation solution containing -15% by weight, rat hepatocytes were cryopreserved for 7 days, and then the survival rate when thawed was examined. The results are shown in Table 3.

【0028】[0028]

【表3】 [Table 3]

【0029】[0029]

【発明の効果】本発明の凍結保存液を用いれば、従来の
DMSOのみ、あるいはDMSOと血清のみを凍結障害
保護剤として添加した保存液より凍結−融解後の細胞の
生存率が上昇した。本発明の凍結保存液は肝実質細胞の
ごとき大型細胞の凍結保存において特に好適に用いられ
る。
EFFECTS OF THE INVENTION Using the cryopreservation liquid of the present invention, the survival rate of cells after freeze-thawing was increased as compared with the conventional preservation liquid containing only DMSO or DMSO and serum alone as a cryoprotective agent. The cryopreservation liquid of the present invention is particularly preferably used for cryopreservation of large cells such as hepatocytes.

Claims (8)

【特許請求の範囲】[Claims] 【請求項1】 ケラチン加水分解物、加水分解ゼラチン
および血清アルブミンからなる群から選択されるタンパ
ク質を0.5重量%以上含有する動物細胞の凍結保存
液。
1. A cryopreservation liquid for animal cells containing 0.5% by weight or more of a protein selected from the group consisting of keratin hydrolyzate, hydrolyzed gelatin and serum albumin.
【請求項2】 ケラチン加水分解物が、可溶化ウールケ
ラチンであって、分子量が10000〜20000であ
る請求項1記載の凍結保存液。
2. The cryopreservation liquid according to claim 1, wherein the keratin hydrolyzate is solubilized wool keratin and has a molecular weight of 10,000 to 20,000.
【請求項3】 ケラチン加水分解物を0.5重量%から
15重量%含有する請求項1または2記載の凍結保存
液。
3. The cryopreservation liquid according to claim 1, which contains 0.5 to 15% by weight of a keratin hydrolyzate.
【請求項4】 加水分解ゼラチンの平均分子量が300
0〜20000である請求項1記載の凍結保存液。
4. The average molecular weight of hydrolyzed gelatin is 300.
The cryopreservation liquid according to claim 1, which is 0 to 20000.
【請求項5】 加水分解ゼラチンを0.5重量%から1
5重量%含有する請求項1または4記載の凍結保存液。
5. Hydrolyzed gelatin from 0.5% by weight to 1
The cryopreservation liquid according to claim 1 or 4, containing 5% by weight.
【請求項6】 血清アルブミンを2.5重量%以上含有
する請求項1記載の凍結保存液。
6. The cryopreservation liquid according to claim 1, which contains 2.5% by weight or more of serum albumin.
【請求項7】 さらにセルロース誘導体およびポリエチ
レングリコールからなる群から選択される高分子を含有
する請求項1から7いずれかに記載の凍結保存液。
7. The cryopreservation liquid according to claim 1, further comprising a polymer selected from the group consisting of cellulose derivatives and polyethylene glycol.
【請求項8】 動物細胞が肝実質細胞である請求項1か
ら8いずれかに記載の凍結保存液。
8. The cryopreservation liquid according to claim 1, wherein the animal cells are liver parenchymal cells.
JP6046859A 1994-03-17 1994-03-17 Frozen storage solution for mammalian cell Pending JPH07255469A (en)

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