-Detailed Information [JCRB0539]-

Notes for the cultivation of human normal diploid cells (JCRB05XX)

Some lots of cell lines registered as JCRB05XX were frozen by using glycerol as cryoprotectant instead of DMSO. The cryoprotectant used was described on data sheet of the cell line. In the case of cells frozen using glycerol, the method for re-cultivation should be as follows.

1. Thaw the ampule contents as quickly as possible in 37 C water bath.

2. Under sterile condition, aspirate the ampule contents (about 1 ml) and transfer them to culture dish (6-cm diameter) or 25cm2 culture flask containing 4 ml culture medium. Do not centrifuge the ampule contents, because the replacement of glycerol is very slow and centrifugation/resuspension causes osmotic shock to the cells.

3. Cultivate the cells in CO2 incubator at 37 C for 12-16 hrs.

4. Exchange the all culture medium by fresh medium.

JCRB05**番代で登録されているヒト正常二倍体細胞の凍結細胞アンプル取り扱い上の注意

ヒト正常細胞は、凍結アンプルを入手してから融解再培養のプロセスで、生存率の低下をおこしやすいとされています。細胞凍結アンプルの温度上昇を極力回避するよう、以下の点にご注意ください。

①細胞凍結アンプルの取り扱い

受領したヒト正常二倍体細胞は、その日のうちに保管容器に移すか融解再培養を行ってください。保管には液体窒素保存容器を推奨します。-80℃超低温槽での長期保管はお勧めしません。保管容器に移す場合は、移動プロセスの間での凍結アンプルの温度上昇が致命的になるので、これを可能な限り回避してください。例えばドライアイスからの取り出しを保管容器のすぐそばで行う、アンプルホルダーを前もって液体窒素で冷却して用いるなどです。

②凍結アンプルの融解再培養

融解再培養する場合は、ドライアイス入り輸送箱、または液体窒素凍結細胞保管容器から取りだした凍結アンプルを温度上昇させることなく（室温においたりすると致命的になります）、前もって用意した温水（約37℃）に素早くつけて、振りながら融解してください。

③JCRB05**番台の細胞には凍結保護剤としてDMSOを用いて凍結しているものとglycerolを用いて凍結しているものがあります（添付のデータシートに記載しています）。この二つでは融解・再培養の方法が異なります。

(1)DMSOを用いた場合の凍結細胞の融解・再培養

1. 凍結保存容器から取りだしたアンプルを、直ちに37℃-40℃の温水にいれて、軽く振りながら素早く解凍する（このステップが最も重要）。この際、ガラスアンプルの破裂の危険があるので、防護面や厚手の保護手袋を装着し、顔や首、手を防護すること。

2. ガラスアンプルを70%エタノールで消毒し、開封する。(アンプルのネックにはあらかじめヤスリが入っています)

3. 融解後、アンプルの内容（約1mlです）をパスツールピペットなどで吸いとり、9mlの冷やした培養液に加えて、1000 rpm，5 min 遠心する。細胞沈殿を5mlの培養液に浮遊し、6センチシャーレ一枚、または25cm2 フラスコ一本にいれて再培養を行う。(過度の希釈は増殖、生存率の低下を招きます)

(2)glycerolを用いた場合の凍結細胞の融解・再培養

1.2.はDMSOの場合と同じ手順で行う。

3. 融解後、4mlの培養液の入った培養ディッシュ（6センチシャーレ、または25cm2 フラスコ）に解凍したアンプルの内容を入れて炭酸ガスインキュベーターに静置する。翌日培地交換する。
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JCRB No.	JCRB0539	Cell Name	TIG-113
Profile	Human normal fibroblast from skin.	Other Name
Animal	human	Strain
Genus	Homo	Species	sapiens
Sex	F	Age	21-year-old
Identity	available	Tissue for Primary Cancer	skin (precordium)
Case history	normal	Metastasis
Tissue Metastasized		Genetics	2n=46, XX
Life Span	finite	Crisis PDL
Morphology	fibroblast-like	Character	Maximum PD = 53. Cells may be distributed around PD15-PD20.
Classify	normal	Established by	Tokyo Metropolitan Institute of Gerontrogy (TMIG)
Registered by	TMIG	Regulation for Distribution
Comment		Year	2003
Medium	Eagle's minimal essential medium with 10% fetal bovine serum.	Methods for Passages	Wash in CMF-PBS.Add 0.25% trypsin. Incubate at 37C for 10-15min. Suspend cells in medium.
Cell Number on Passage	5 x 10^3 cells/sq.cm.	Race	Japanese
CO2 Conc.		Tissue Sampling	skin
Tissue Type

Detection of virus genome fragment by Real-time PCR
Detected DNA Virus	tested	Detected RNA Virus	tested
CMV	-	parvoB19	-	HCV	-	HTLV-1	-
EBV	-	HBV	-	HIV-1	-	HTLV-2	-
HHV6	-	HTLV-1	-	HIV-2	-	HAV	-
HHV7	-	HTLV-2	-	-/negative. +/positive. nt/not tested. (positive (+) does not immediately mean the production of infectious viral particles.)
BKV	-	HIV-1	-
JCV	-	HIV-2	-
ADV	-	HPV18	-
Notes

Link
Cellosaurus(ExPASy) Cellosaurus (ExPASy) is developed by Amos Bairoch of the CALIPHO group at the SIB - Swiss Institute of Bioinformatics as part of the neXtProt project. Cellosaurus is a knowledge database of cell lines with various information summarized.	CVCL_3198

Reference
Pubmed id:7556460	Fetal-adult phenotype transition, in terms of the serum dependency and growth factor requirements, of human skin fibroblast migration. Kondo H,Yonezawa Y Exp Cell Res. 1995 Oct;220(2):501-4

01282013
04162008
11082018

Cell No.	JCRB0539	Cell Name
LOT No.	01282013	Lot Specification	distribution
Medium	Eagle's minimum essential medium with 10% fetal bovine serum. This lot was frozen using DMSO.	Temperature	37 C
Cell Density at Seeding	2.7 - 3.2 x 10^4 cells/mL	Methods for Passages	Cells were harvested after treatment with 0.25% trypsin and 0.02% EDTA.
Doubling Time	approx. 41 hrs.	Cell Number in Vial (cells/1ml)	3.0 x 10^5
Viability at cell freezing (%)	75.0	Antibiotics Used	free
Passage Number	Unknown (3 at bank)	PDL	25.2
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - EMEM	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0539	Cell Name
LOT No.	04162008	Lot Specification	distribution
Medium	Eagle's minimum essential medium with 10% fetal bovine serum (FBS Lot; GIBCO Cat. No. 10099, Lot 484375). This lot of ampoule was frozen using DMSO.	Temperature	37 C
Cell Density at Seeding	2 - 3 x 10^4 cells/ml	Methods for Passages	0.25% trypsin and 0.02% EDTA. Split ratio 1:4 is recommended.
Doubling Time	1.7 days	Cell Number in Vial (cells/1ml)	6.0 x 10^5
Viability at cell freezing (%)	77.3	Antibiotics Used	free
Passage Number	Unknown (3 at bank)	PDL	23
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	Confirmed as human by NP, G6PD (type B), MD.
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - EMEM	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0539	Cell Name	TIG-113
LOT No.	11082018	Lot Specification	distribution
Medium	Eagle's minimum essential medium with 10% fetal bovine seum (FBS; Sigma Cat. # 172012)	Temperature	37 C
Cell Density at Seeding	3.8 - 6.0 x 10^4 cells/mL	Methods for Passages	Cells were harvested after treatment with 0.25% trypsin and 0.02% EDTA.
Doubling Time	approx. 45 hrs.	Cell Number in Vial (cells/1ml)	5.7 x 10^5
Viability at cell freezing (%)	88.9	Antibiotics Used	free
Passage Number	Unknown (3 at bank)	PDL	22.9
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - EMEM	CO2 Conc.	5%
Viability immediately after thawing (%)	91.1	Additional information

JCRB Cell Bank

JCRB0539 TIG-113

Cell information

LOT Information