JPH058675B2 - - Google Patents
Info
- Publication number
- JPH058675B2 JPH058675B2 JP62050950A JP5095087A JPH058675B2 JP H058675 B2 JPH058675 B2 JP H058675B2 JP 62050950 A JP62050950 A JP 62050950A JP 5095087 A JP5095087 A JP 5095087A JP H058675 B2 JPH058675 B2 JP H058675B2
- Authority
- JP
- Japan
- Prior art keywords
- medium
- serum
- cells
- free
- cryopreservation
- 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.)
- Expired - Lifetime
Links
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 32
- 239000012595 freezing medium Substances 0.000 claims description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 12
- 229920000609 methyl cellulose Polymers 0.000 claims description 12
- 239000001923 methylcellulose Substances 0.000 claims description 12
- 235000011187 glycerol Nutrition 0.000 claims description 6
- 239000002577 cryoprotective agent Substances 0.000 claims description 3
- LNAZSHAWQACDHT-XIYTZBAFSA-N (2r,3r,4s,5r,6s)-4,5-dimethoxy-2-(methoxymethyl)-3-[(2s,3r,4s,5r,6r)-3,4,5-trimethoxy-6-(methoxymethyl)oxan-2-yl]oxy-6-[(2r,3r,4s,5r,6r)-4,5,6-trimethoxy-2-(methoxymethyl)oxan-3-yl]oxyoxane Chemical compound CO[C@@H]1[C@@H](OC)[C@H](OC)[C@@H](COC)O[C@H]1O[C@H]1[C@H](OC)[C@@H](OC)[C@H](O[C@H]2[C@@H]([C@@H](OC)[C@H](OC)O[C@@H]2COC)OC)O[C@@H]1COC LNAZSHAWQACDHT-XIYTZBAFSA-N 0.000 claims description 2
- 235000010981 methylcellulose Nutrition 0.000 claims 2
- 210000004027 cell Anatomy 0.000 description 30
- 239000002609 medium Substances 0.000 description 20
- 210000004102 animal cell Anatomy 0.000 description 8
- 238000007710 freezing Methods 0.000 description 7
- 230000008014 freezing Effects 0.000 description 7
- 238000005138 cryopreservation Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 2
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000007758 minimum essential medium Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000012679 serum free medium Substances 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 206010025323 Lymphomas Diseases 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- MKXKFYHWDHIYRV-UHFFFAOYSA-N flutamide Chemical compound CC(C)C(=O)NC1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 MKXKFYHWDHIYRV-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
〔産業上の利用分野〕
本発明は、培養動物細胞を凍結状態で長期間保
存する際に必要な凍結保存用培地、特に血清を含
まない培地に関する。
〔従来の技術〕
培養動物細胞は、一般に継代培養中に諸種の性
質が変化しやすい。その変化は、細胞増殖能、物
質生産能、染色体数等にあらわれる。このような
細胞の性質の変化をできる限り回避し、安定した
性質を示す細胞を繰り返し、再現性よく使用する
ために、凍結状態で細胞を長期間保存する技術
は、学研研究、商業目的を問わず不可欠である。
このために使用される細胞凍結保存用の培地とし
ては、現在凍結障害防護剤としてグリセリン
（glycerin）またはジメチルスルホキシド
（dimethylsulfoxide、以下DMSOという）、並び
に血清を含む培地が広く使用されている。
しかしながら、培養動物細胞の中には、血清を
全く含まない培地で培養されているものがあり、
この無血清培養細胞の凍結保存のためには、血清
を含まない凍結保用存培地知の開発が望まれてい
る。
〔発明が解決しようとする問題点〕
したがつて本発明の目的は、無血清培養動物細
胞の凍結保存に適した、血清を含まない凍結保存
用培地を提供することである。
〔問題点を解決するための手段〕
上記目的は、凍結障害防護剤として、メチルセ
ルロースと、ジメチルスルホキシドまたはグリセ
リンとを含む無血清凍結保存用培地により達成さ
れる。
本発明の無血清凍結保存用培地は下記の成分を
含んでいる。
(イ) 培養動物細胞の種類に応じて使用される培地
から血清を除いたもの。これは一般に合成培地
と称される。例えば、イーグルの最少必須培地
（Eagle′s minimun essential medium）（以下
MEM培地という）、ハムのＦ−12培地
（Ham'sF−12medium）、RPMI1640培地、
DM−160培地、三橋の培地等である。
(ロ) メチルセルロース
(ハ) DMSOまたはグリセリン
メチルセルロースの含有量は、好ましくは0.02
〜１％（ｗ／ｖ）、さらに好ましくは0.05〜0.2％
（ｗ／ｖ）である。0.02％より少ないときは、メ
チルセルロースによる凍結障害防護効果が不十分
となり、１％を越えると、凍結保存培地の粘性が
高くなりすぎて、実用的見地から好ましくない。
DMSOまたはグリセリンの含有量は、好まし
くは１〜20％（ｖ／ｖ）、さらに好ましくは５〜
15％（ｖ／ｖ）である。１％以下では、基本的な
凍結障害防護効果があらわれず、20％を越える
と、直接的な能細胞障害作用を示すようになり好
ましくない。
本発明の無血清凍結保存用培地は、たとえば以
下のようにして使用される。
(i) 培養細胞を無血清凍結保存用培地に懸濁す
る。または培養基質に付着した状態の細胞が接
する培地を無血清凍結保存用培地に交換する。
(ii) −70℃以下で凍結保存する
本発明の無血清凍結保存用培地は、すべての
培養動物細胞を長期間凍結保存することができ
るが、このような動物細胞の例としては、Ｌ・
P3細胞、ハイブリドーマ193、HeLa・P3（ヒト
子宮癌由来）、L5178Y（マウスリンパ腫細胞）
などがあげられる。
以下実施例により本発明をさらに具体的に説明
する。
実施例 １
Ｌ・P3細胞の凍結保存
(1) ２ｇのメチルセルロースを100mlの純水に懸
濁し、オートクレーブで滅菌後、４℃で一夜撹
拌、溶解する。
(2) 通常使用されるDM−160培地の溶質を２倍
濃度に調整したDM−160培地１容と(1)のメチ
ルセルロース液１容を混ぜ、さらにこの混合液
に通常のDM−160培地６容とDMSO2容を加え
る。これを２倍濃度無血清凍結保存用培地とす
る。
(3) 培養されているＬ・P3細胞の培地を除き、
スクレーパーにより細胞をはがす。
(4) 軽くピペツテイングを行い、細胞を分散させ
る。
(5) 細胞を1000rmp、３分間の遠心分離によつて
収集する。
(6) 収集した細胞を通常のDM−160培地で１回
洗う。
(7) この細胞を通常のDM−160培地に懸濁し、
細胞密度を２×106個／ml前後に合わせる。
(8) (2)で調整した２倍濃度無血清凍結保存用培地
を同量、または1/3量添加し、よく混合する。
(9) 凍結用アンプルに１mlづつ分注し、プログラ
ムフリーザーに入れ、−１℃／minの速度で−
70℃以下にまで冷却し、凍結する。
(10) 超低温フリーザーまたは凍結保存用液体窒素
タンクに保存する。
この方法によつて24時間凍結保存されたＬ・
P3細胞の生存率は表１に示されている。この細
胞は、通常の無血清培養で対数増殖期にあつて
も、若干の死細胞を細胞集団中に含んでいる性質
があるが、その細胞集団中の凍結前の生存率はメ
チルセルロースによる影響を受けない。メチルセ
ルロースを含まない凍結保存用培地（control）
に比べ、上記の無血清凍結保存用培地を用いた場
合は、凍結融解後も1.7倍以上の生存率を保持し
ている。
[Industrial Application Field] The present invention relates to a cryopreservation medium necessary for preserving cultured animal cells in a frozen state for a long period of time, particularly a serum-free medium. [Prior Art] Generally, various properties of cultured animal cells tend to change during subculture. The changes appear in cell proliferation ability, substance production ability, chromosome number, etc. In order to avoid such changes in cell properties as much as possible and to repeatedly and reproducibly use cells that exhibit stable properties, the technology of storing cells in a frozen state for long periods of time is required for both academic research and commercial purposes. It is essential.
As a cell cryopreservation medium used for this purpose, a medium containing glycerin or dimethylsulfoxide (hereinafter referred to as DMSO) as a cryoprotective agent and serum is currently widely used. However, some cultured animal cells are cultured in media that do not contain serum at all.
In order to cryopreserve serum-free cultured cells, it is desired to develop a cryopreservation medium that does not contain serum. [Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a serum-free cryopreservation medium suitable for cryopreservation of serum-free cultured animal cells. [Means for Solving the Problems] The above object is achieved by a serum-free cryopreservation medium containing methylcellulose and dimethyl sulfoxide or glycerin as a cryoprotectant. The serum-free cryopreservation medium of the present invention contains the following components. (b) A culture medium that is used depending on the type of cultured animal cells, with serum removed. This is commonly referred to as a synthetic medium. For example, Eagle's minimum essential medium (hereinafter referred to as Eagle's minimum essential medium)
MEM medium), Ham's F-12 medium, RPMI1640 medium,
These include DM-160 medium and Mitsuhashi medium. (b) Methylcellulose (c) DMSO or glycerin The content of methylcellulose is preferably 0.02
~1% (w/v), more preferably 0.05-0.2%
(w/v). If it is less than 0.02%, the effect of protecting against freezing damage by methylcellulose will be insufficient, and if it exceeds 1%, the viscosity of the cryopreservation medium will become too high, which is undesirable from a practical standpoint. The content of DMSO or glycerin is preferably 1 to 20% (v/v), more preferably 5 to 20% (v/v).
15% (v/v). If it is less than 1%, the basic effect of protecting against freezing damage will not be exhibited, and if it exceeds 20%, it will show a direct cytotoxic effect, which is not preferable. The serum-free cryopreservation medium of the present invention is used, for example, as follows. (i) Suspend cultured cells in serum-free cryopreservation medium. Alternatively, the medium in contact with the cells attached to the culture substrate is replaced with a serum-free cryopreservation medium. (ii) Cryopreservation at −70°C or below The serum-free cryopreservation medium of the present invention can freeze-preserve all cultured animal cells for a long period of time. Examples of such animal cells include L.
P3 cells, hybridoma 193, HeLa・P3 (derived from human uterine cancer), L5178Y (mouse lymphoma cells)
etc. can be mentioned. The present invention will be explained in more detail with reference to Examples below. Example 1 Cryopreservation of L/P3 cells (1) 2 g of methylcellulose is suspended in 100 ml of pure water, sterilized in an autoclave, and then stirred and dissolved overnight at 4°C. (2) Mix 1 volume of DM-160 medium, in which the solutes of the commonly used DM-160 medium have been adjusted to twice the concentration, and 1 volume of the methylcellulose solution from (1), and then add 6 volumes of the usual DM-160 medium to this mixture. Add 2 volumes and 2 volumes of DMSO. This is used as a double concentration serum-free cryopreservation medium. (3) Except for the culture medium of L/P3 cells,
Peel off the cells with a scraper. (4) Gently pipette to disperse the cells. (5) Collect cells by centrifugation at 1000 rpm for 3 minutes. (6) Wash the collected cells once with regular DM-160 medium. (7) Suspend these cells in regular DM-160 medium,
Adjust the cell density to around 2×10 6 cells/ml. (8) Add the same volume or 1/3 volume of the double concentration serum-free cryopreservation medium prepared in (2) and mix well. (9) Dispense 1 ml into freezing ampoules, place in a program freezer, and incubate at a rate of -1℃/min.
Cool to below 70℃ and freeze. (10) Store in an ultra-low temperature freezer or liquid nitrogen tank for cryopreservation. L.
The viability of P3 cells is shown in Table 1. These cells have the property of containing some dead cells in the cell population even in the logarithmic growth phase in normal serum-free culture, but the survival rate of the cell population before freezing is not affected by methylcellulose. I don't accept it. Cryopreservation medium without methylcellulose (control)
Compared to this, when the above serum-free cryopreservation medium was used, the survival rate was 1.7 times higher even after freezing and thawing.
【表】
最終細胞密度２×106／mlで凍結し、24時間後
解凍した。解凍後、細胞を通常の培地で一回洗浄
し、0.06％のトリパンブルーを含む通常の培地を
加え、２分間放置した。その後、リン酸緩衝生理
食塩水で一度洗浄し、血球計算盤で総細胞数と、
トリパンブルーで染色されなかつた生細胞を計数
し、生存率を産出した。
対照は10％のDMSOのみを含むDM−160培地、
No.１は0.1％メチルセルロースと10％DMSOを含
むDM−160培地、No.２は0.05％メチルセルロー
スと５％DMSOを含むDM−160培地である。
実施例 ２
ハイブリドーマ193−(1)の凍結保存
(1) 実施例１の(1)、(2)に従つて２倍濃度無血清凍
結保存用培地を作製する。但しDM−160培地
の代りにMEM培地を使用する。
(2) この２倍濃度無血清凍結保存用培地１容と、
無血清培地SFM101で培養されているハイブリ
ドーマ193−(1)細胞浮遊液１容を混合する。
(3) 凍結用アンプルに１mlづつ分注し、プログラ
ムフリーザーに入れ、−１℃／minの速度で−
70℃以下にまで冷却し、凍結する。
(10) 超低温フリーザーまたは凍結保存用液体窒素
タンクに保存する。
この方法によつて24時間凍結保存された細胞の
回収率、生存率は表２に示されている。この細胞
は凍結しなくても10％DMSOを含む培地に懸濁
しただけで生存率が低下する傾向を示す性質があ
るが、凍結融解後は、メチルセルロースを含まな
い凍結保存用培地（対照）に比べ、上記の無血清
凍結保存用培地を用いた場合は、凍結融解後も10
％血清添加凍結保存用培地とほぼ同等の細胞回収
率、生存率を保持している。[Table] The cells were frozen at a final cell density of 2×10 6 /ml and thawed after 24 hours. After thawing, the cells were washed once with normal medium, and normal medium containing 0.06% trypan blue was added and left for 2 minutes. Afterwards, the cells were washed once with phosphate buffered saline, and the total cell count was determined using a hemocytometer.
Live cells that were not stained with trypan blue were counted and the viability was calculated. Control is DM-160 medium containing only 10% DMSO;
No. 1 is a DM-160 medium containing 0.1% methylcellulose and 10% DMSO, and No. 2 is a DM-160 medium containing 0.05% methylcellulose and 5% DMSO. Example 2 Cryopreservation of Hybridoma 193-(1) (1) A double concentration serum-free cryopreservation medium is prepared according to (1) and (2) of Example 1. However, MEM medium is used instead of DM-160 medium. (2) 1 volume of this double concentration serum-free cryopreservation medium,
Mix 1 volume of hybridoma 193-(1) cell suspension cultured in serum-free medium SFM101. (3) Dispense 1 ml each into freezing ampoules, place in a program freezer, and freeze at a rate of -1℃/min.
Cool to below 70℃ and freeze. (10) Store in an ultra-low temperature freezer or liquid nitrogen tank for cryopreservation. The recovery rate and survival rate of cells cryopreserved for 24 hours by this method are shown in Table 2. Even if these cells are not frozen, their survival rate tends to decrease just by suspending them in a medium containing 10% DMSO. In comparison, when using the serum-free cryopreservation medium mentioned above, even after freezing and thawing,
% serum-added cryopreservation medium, maintaining cell recovery rate and survival rate almost the same.
本発明の無血清凍結保存用培地によれば、血清
を全く含まない培地で培養されている無血清培養
動物細胞を、凍結状態で長期間安定に保存するこ
とができる。
According to the serum-free cryopreservation medium of the present invention, serum-free cultured animal cells that are cultured in a medium that does not contain any serum can be stably stored in a frozen state for a long period of time.
Claims (1)
と、ジメチルスルホキシドまたはグリセリンとを
含むことを特徴とする無血清凍結保存用培地。 ２ メチルセルロースの含有量が、0.02〜1.0％
（ｗ／ｖ）である特許請求の範囲第１項記載の無
血清凍結保存用培地。 ３ ジメチルスルホキシドまたはグリセリンの含
有量が、１〜20％（ｖ／ｖ）である特許請求の範
囲第１項または第２項記載の無血清凍結保存用培
地。[Scope of Claims] 1. A serum-free cryopreservation medium characterized by containing methylcellulose and dimethyl sulfoxide or glycerin as a cryoprotectant. 2 Methyl cellulose content is 0.02-1.0%
(w/v) The serum-free cryopreservation medium according to claim 1. 3. The serum-free cryopreservation medium according to claim 1 or 2, wherein the content of dimethyl sulfoxide or glycerin is 1 to 20% (v/v).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62050950A JPS63216476A (en) | 1987-03-05 | 1987-03-05 | Serum-free medium for freeze stocking |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62050950A JPS63216476A (en) | 1987-03-05 | 1987-03-05 | Serum-free medium for freeze stocking |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63216476A JPS63216476A (en) | 1988-09-08 |
| JPH058675B2 true JPH058675B2 (en) | 1993-02-02 |
Family
ID=12873102
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62050950A Granted JPS63216476A (en) | 1987-03-05 | 1987-03-05 | Serum-free medium for freeze stocking |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS63216476A (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0646840A (en) * | 1992-08-04 | 1994-02-22 | Nippon Zenyaku Kogyo Kk | Solution for freezing and storing cell |
| ATE426016T1 (en) * | 1999-09-24 | 2009-04-15 | Cybios Llc | PLURIPOTENT EMBRYONAL STEM CELL-LIKE CELLS, COMPOSITIONS AND USES THEIR |
| ATE315080T1 (en) | 2000-06-28 | 2006-02-15 | Ascendia Ab | DNA/RNA EMBEDING MEDIUM AND METHOD OF USE |
| US8460926B2 (en) | 2005-11-17 | 2013-06-11 | Nippon Zenyaku Kogyo Co., Ltd | Aqueous solution for cell preservation |
| JP5238501B2 (en) * | 2006-07-12 | 2013-07-17 | 日本全薬工業株式会社 | Semen diluted storage composition |
| NO331476B1 (en) * | 2007-12-21 | 2012-01-16 | Jaffar Ali Bin M Abdullah | Protein-free gamete and embryo handling and culture media products containing methyl cellulose |
-
1987
- 1987-03-05 JP JP62050950A patent/JPS63216476A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63216476A (en) | 1988-09-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jang et al. | Cryopreservation and its clinical applications | |
| CN108207930A (en) | A kind of cocktail type cryoprotector and its application | |
| CN109221082A (en) | Cells frozen storing liquid, cryopreservation resuscitation method and its application | |
| Sasnoor et al. | Supplementation of conventional freezing medium with a combination of catalase and trehalose results in better protection of surface molecules and functionality of hematopoietic cells | |
| JP2000344602A (en) | Preservative for animal cell or organ and its preservation | |
| CN108651439A (en) | A kind of complete serum-free frozen stock solution of peripheral blood mononuclear cells and its method | |
| JP6353615B1 (en) | Mammalian cell cryopreservation solution | |
| CN109329271A (en) | A kind of clinical mescenchymal stem cell freezen protective liquid and preparation method thereof | |
| WO2014038473A1 (en) | Method for maintaining organ or tissue for transplantation use for long period | |
| JP2006230396A (en) | Cell preservative liquid | |
| CN105941389A (en) | Animal derived serum-free cell freezing medium | |
| Stoll et al. | Membrane stability during biopreservation of blood cells | |
| Wowk | How cryoprotectants work | |
| CN103704206A (en) | Placenta preserving fluid | |
| CN106942203B (en) | IPS cell preservation solution and preparation method thereof | |
| JP2001247401A (en) | Cooling preservation liquid for tissue | |
| KR20090059289A (en) | The preservation method of peripheral blood mononuclear cells at high density condition | |
| JPH058675B2 (en) | ||
| CN116473051A (en) | Serum-free non-program cell frozen stock solution and preparation method and application thereof | |
| US5895745A (en) | Method of thawing cryopreserved cells | |
| Brockbank et al. | Storage of tissues by vitrification | |
| CN107711823B (en) | Cell cryopreservation liquid stored at normal temperature and application thereof | |
| CN108029679B (en) | Freezing medium for freezing mononuclear cells | |
| Hunt et al. | Optimising cryopreservation protocols for haematopoietic progenitor cells: a methodological approach for umbilical cord blood | |
| JP4385158B2 (en) | Cell preservation solution and cell preservation method using the preservation solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| EXPY | Cancellation because of completion of term |