-Detailed Information [JCRB0521]-

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.	JCRB0521	Cell Name	MRC-5-30
Profile	fetal lung, normal diploid fibroblast, PDL=about 30	Other Name
Animal	human	Strain
Genus	Homo	Species	sapiens
Sex	M	Age	14 weeks
Identity	available	Tissue for Primary Cancer	lung
Case history		Metastasis
Tissue Metastasized		Genetics	lung,normal diploid fibroblast
Life Span		Crisis PDL
Morphology		Character	lung,normal diploid fibroblast
Classify	normal	Established by	Jacobs,J.P. et al.
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.Incubation at 37C for 10-15 min.Suspend cells in culture medium.
Cell Number on Passage	sprit ratio=1/4	Race
CO2 Conc.	5 %	Tissue Sampling	fetus 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_3027

Reference
Pubmed id:7389832	A new human diploid cell strain, TIG-1, for the research on cellular aging. Ohashi M,Aizawa S,Ooka H,Ohsawa T,Kaji K,Kondo H,Kobayashi T,Noumura T,Matsuo M,Mitsui Y,Murota S,Yamamoto K,Ito H,Shimada H,Utakoji T Exp Gerontol. 1980;15(2):121-33
Pubmed id:12635218	Error theory and ageing in human diploid fibroblasts. Lewis CM,Tarrant GM Nature. 1972 Oct 6;239(5371):316-8
Pubmed id:12635262	Altered enzymes in ageing human fibroblasts. Holliday R,Tarrant GM Nature. 1972 Jul 7;238(5358):26-30
Pubmed id:4316953	Characteristics of a human diploid cell designated MRC-5. Jacobs JP,Jones CM,Baille JP Nature. 1970 Jul 11;227(5254):168-70

05142008
R052
10072013
09112017
05172021

Cell No.	JCRB0521	Cell Name
LOT No.	05142008	Lot Specification	distribution
Medium	Eagle's minimum essential medium with 10% fetal bovine serum (GIBCO Cat.# 10099, lot 484375). This lot was frozen using DMSO.	Temperature	37 C
Cell Density at Seeding	4 - 8 x 10^4 cells/ml	Methods for Passages	0.25% trypsin and 0.02% EDTA.
Doubling Time	approx. 40 hrs	Cell Number in Vial (cells/1ml)	2.3 x 10^6
Viability at cell freezing (%)	94.5	Antibiotics Used	free
Passage Number	Unknown (3 at bank)	PDL	29.5
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.	JCRB0521	Cell Name
LOT No.	R052	Lot Specification	distribution
Medium	Eagle's minimal essential medium with 10% FCS This stock was freezed using DMSO.	Temperature	37 C
Cell Density at Seeding		Methods for Passages	Treat cells with 0.25% trypsin for 10-15 min. Split ratio, 1:4.
Doubling Time		Cell Number in Vial (cells/1ml)	1.2 x 10^6
Viability at cell freezing (%)	90.0	Antibiotics Used	100 ug/ml streptmycin and 100 units/ml penicillin
Passage Number		PDL	29
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing		CO2 Conc.
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0521	Cell Name	MRC-5-30
LOT No.	10072013	Lot Specification	distribution
Medium	Eagle's minimum essential medium with 10% fetal bovine serum (FBS; GIBCO Cat. #10099). This lot was frozen using DMSO.	Temperature	37C
Cell Density at Seeding	sprit ratio=1/4 (3.3 - 15 x 10^4 cells/mL)	Methods for Passages	Wash in CMF-PBS.Add 0.25% trypsin - 0.02% EDTA. Incubation at 37C for 10-15 min.Suspend cells in culture medium.
Doubling Time		Cell Number in Vial (cells/1ml)	1.3 x 10^6 (cells/1m
Viability at cell freezing (%)		Antibiotics Used	free
Passage Number	Unknown (3 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	G6PD(typeB),NP,LDH were examined. Human,not HeLa
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion		Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS -EMEM	CO2 Conc.	5%
Viability immediately after thawing (%)	93.0%	Additional information

Cell No.	JCRB0521	Cell Name	MRC-5-30
LOT No.	09112017	Lot Specification	distribution
Medium	Eagle's minimum essential medium with 10% fetal bovine serum (FBS; Sigma Cat. # 172012)	Temperature	37 C
Cell Density at Seeding	5.2 - 5.8 x 10^4 cells/mL	Methods for Passages	Cells were harvested after treatment with 0.25% trypsin and 0.02% EDTA.
Doubling Time	approx. 26 hrs.	Cell Number in Vial (cells/1ml)	1.0 x 10^6
Viability at cell freezing (%)	89.5	Antibiotics Used	free
Passage Number	Unknown (3 at bank)	PDL	PDL=27.2
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 (%)	89.5	Additional information

Cell No.	JCRB0521	Cell Name	MRC-5-30
LOT No.	05172021	Lot Specification	distribution
Medium	Eagle's minimum essential medium with 10% fetal bovine serum (FBS; Sigma Cat. # 172012)	Temperature	37 C
Cell Density at Seeding	2.7 x 10^4 cells/mL	Methods for Passages	Cells were harvested after treatment with 0.25% trypsin and 0.02% EDTA.
Doubling Time	approx. 32 hrs	Cell Number in Vial (cells/1ml)	7.8 x 10^5
Viability at cell freezing (%)	99	Antibiotics Used	free
Passage Number	Unknown (2 at bank)	PDL	29
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	Additional information

JCRB Cell Bank

JCRB0521 MRC-5-30

Cell information

LOT Information