-Detailed Information [JCRB0518]-

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.	JCRB0518	Cell Name	WI-38-40
Profile	Normal primary cells from lung as well as JCRB0517. Now only the JCRB0518 with PDL 40 is available.	Other Name
Animal	human	Strain
Genus	Homo	Species	sapiens
Sex	F	Age
Identity	available	Tissue for Primary Cancer	lung
Case history	no	Metastasis
Tissue Metastasized		Genetics	lung, normal diploid fibroblast
Life Span		Crisis PDL
Morphology		Character	lung, normal diploid fibroblast
Classify	new	Established by	Hayflick,L.
Registered by	Ooka,H.	Regulation for Distribution
Comment		Year	1989
Medium	Eagle's minimal essential medium with 10% fetal bovine serum	Methods for Passages	Wash in CMF-PBS.Add 0.25% trypsin.Keep at 37C for 10-15 min. Suspend cells in medium.
Cell Number on Passage	split ratio=1/4	Race	Caucasian
CO2 Conc.	5 %	Tissue Sampling	lung
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_2813

Reference
Pubmed id:4290661	SV40 transformation of human diploid cells. A parallel study of viral and karyologic parameters. Girardi AJ,Weinstein D,Moorhead PS Ann Med Exp Biol Fenn. 1966;44(2):242-54
Pubmed id:14315085	THE LIMITED IN VITRO LIFETIME OF HUMAN DIPLOID CELL STRAINS. HAYFLICK L Exp Cell Res. 1965 Mar;37():614-36
Pubmed id:14288316	SV40-INDUCED TRANFORMATION OF HUMAN DIPLOID CELLS: CRISIS AND RECOVERY. GIRARDI AJ,JENSEN FC,KOPROWSKI H J Cell Comp Physiol. 1965 Feb;65():69-83
Pubmed id:13905660	Preparation of poliovirus vaccines in a human fetal diploid cell strain. HAYFLICK L,PLOTKIN SA,NORTON TW,KOPROWSKI H Am J Hyg. 1962 Mar;75():240-58

04102007
R381
R382
11022015

Cell No.	JCRB0518	Cell Name
LOT No.	04102007	Lot Specification	distribution
Medium	Eagle's minimal essential medium with 10% fetal bovine serum (FBS lot; GIBCO 484375). This lot of cells was frozen in DMSO-containing medium.	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	approx. 2 days	Cell Number in Vial (cells/1ml)	3.4 x 10^5
Viability at cell freezing (%)	82.1	Antibiotics Used	free
Passage Number	Unknown	PDL	40.8 PD
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	Confirmed as human by G6PD (typeB), NP, LD, 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.	JCRB0518	Cell Name
LOT No.	R381	Lot Specification	distribution
Medium	MEM +10% FBS (this ampule was freezed using DMSO)	Temperature	37
Cell Density at Seeding		Methods for Passages	Wash in CMF-PBS.Add 0.25% trypsin.Keep at 37C for 10-15 min. Suspend cells in medium.
Doubling Time		Cell Number in Vial (cells/1ml)
Viability at cell freezing (%)		Antibiotics Used	penicillin(100 units/ml)/streptomycin(100ug/ml)
Passage Number		PDL	41
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion		Exoteric Gene
Medium for Freezing		CO2 Conc.
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0518	Cell Name
LOT No.	R382	Lot Specification	distribution
Medium	Eagle's MEM with 10% FBS (JCRB No.0517=0518)This stock was freezed using glycerol.	Temperature	37 C
Cell Density at Seeding	split 1:4	Methods for Passages	Wash in CMF-PBS. Add 0.25% trypsin. Keep at 37 C for 10-15 min. Suspend cells in medium.
Doubling Time		Cell Number in Vial (cells/1ml)
Viability at cell freezing (%)		Antibiotics Used	100 ug/ml streptmycin and 100 units/ml penicilline
Passage Number		PDL	PDL40
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion		Exoteric Gene
Medium for Freezing		CO2 Conc.
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0518	Cell Name	WI-38-40
LOT No.	11022015	Lot Specification	distribution
Medium	Eagle's minimum essential medium with 10% fetal bovine serum (FBS; GIBCO Cat. # 10091). This lot was frozen using DMSO.	Temperature	37 C
Cell Density at Seeding	2.0 - 5.3 x 10^4	Methods for Passages	Cells were harvested after treatment with 0.25% trypsin and 0.02% EDTA.
Doubling Time	36 - 42 hrs.	Cell Number in Vial (cells/1ml)	1.9 x 10^6
Viability at cell freezing (%)	80.5	Antibiotics Used	free
Passage Number	Unknown (3 at bank)	PDL	40
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 (%)	80.5	Additional information

JCRB Cell Bank

JCRB0518 WI-38-40

Cell information

LOT Information