-Detailed Information [JCRB0141]-

JCRB0141 PHK16-0b

Cell information

Important Notice(s)

Supplements for the Serum free culture medium

Supplemented growth hormones for the MCDB153 medium. 

Yasumoto,S. Kanagawa Cancer Center Research Institute. 

[Supplemented growth hormones for the MCDB153 medium.]

5 ug/ml Inslin(1% CH3COOH-DDW) 
0.5ug/ml Hidrocortisone(EtOH) 
10ug/ml transferrin(PBS) 
0.1mM phosphorylethanolamine(DDW) 
0.1mM ethanolamine(DDW) 
10ng/ml EGF(0.1% BSA-PBS) 
40-70ug/ml BPE(PBS) 

[Reference]
 
Jikken Igaku, Supplement
 BioManual Up series (2nd Edition)
 "New Cell Culture methods for Molecular Biology"
 T.Kuroki, N.Hoh and K.Chida
 Yohdo-sha,Co.
 Section 4. Primary cell culture, 2.Keratinocyte P71-80

[Medium]
MCDB153medium Powder (SIGMA cat.No.M-7403) for 1L and adjust to pH7.5 by adding Sodium Bicarbonate 1.176g(1N-NaOH).

MCDB153medium liquid (Functional Peptide Laboratory Inc., IFP) 1L and adust pH to 7.5 by adding stock solution of the sodium bicarbonate (x100-x1000).

[Working solution]
 
Store the working solution in the refregerator up to one month when supplements were added to the medium and only two weeks can be stored in the refregerator when BPE was added.

-- Medium -- 
MCDB153medium liquid (Functional Peptide Laboratory Inc., IFP) 1L and adjust pH to 7.5 by adding stock solution of the sodium bicarbonate (x100-x1000). 

-- Supplement--
Insulin(SIGMA cat.No.I-1882): 5mg/L (1000 x stock solution is prepared in 1% CH3COOH)
Hydrocortisone (SIGMA cat.No.H-4001): 0.5mg/L (10000 x stock solution is prepared in EtOH) 
Transferin (SIGMA cat.No.T8158): 10mg/L (1000 x stock solution is prepared in PBS)  
Ethanolamine (SIGMA cat.No.E-0135): 6.1mg/L (6 ul/L) 
Phosohorylethanolamine (SIGMA P-0503): 14.1mg/L 
EGF (SIGMA cat.No.E-4127): 10ug/L  (1000 x stock solution is prepared in 0.1% BSA-PBS )
BPE(Kyokuto Pherm. Co. cat.No.202000): 40-70mg/L (100 x stock solution is prepared in PBS)  

[Subculture] 

Trypsin solution: Trypsin typeIII(SIGMA cat.No.T-8253) 0.1%  0.02% EDTA in PBS- 
(SIGMA T-8253 was discontinued. Instead, Fujifilm Wako Cat. # 201-19181 will be adequate. Conventional 0.25% trypsin solution will also work.)
Trypsin Inhibitor solution: Trypsin Inhibitor(SIGMA cat.No.T-6522) 2mg/ml in PBS- 
 


[Close]

Cell type：general cells (View Pricing Information)

JCRB No.	JCRB0141	Cell Name	PHK16-0b
Profile	Normal keratinocyte cells immortalized with HPV16 virus genome and the growth is dependent to EGF. More than 95% of cells are diploid.	Other Name
Animal	human	Strain
Genus	Homo	Species	sapiens
Sex	M	Age
Identity	available	Tissue for Primary Cancer	skin
Case history	normal keratinocyte cells immortalized with HPV16.	Metastasis	No
Tissue Metastasized		Genetics	E6/E7 expressed and telomerase positive.
Life Span	infinite	Crisis PDL
Morphology	keratinocyte	Character	Cells immortalized with HPV16 and the growth is dependent to EGF. Over 95% cells are diploid.
Classify	immortalized	Established by	Yasumoto,S.
Registered by	Yasumoto,S.	Regulation for Distribution	Not defined.
Comment	Need details of the culture method. STR-PCR data showed the same pattern with NCC16(NCC16 may be cross contaminated and will be replaced to NCC16-P11).	Year	1999
Medium	MCDB153 medium (0.03 mM Ca2+) with 5 ug/ml insulin, 0.5 ug/ml hydrocortisone, 10 ug/ml transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 10 ng/ml EGF and 40-70 ug/ml BPE. MCDB153 with < 0.5% FCS was also used in original report.	Methods for Passages	Cells are harvested with 0.1% trypsin and 0.01% EDTA. Medium change every 2 days and subculture every 10 days.
Cell Number on Passage	1.5 - 2.4 x 10^5 cells/sq.cm.	Race	Caucasian
CO2 Conc.	5 %	Tissue Sampling	foreskin
Tissue Type

Detection of virus genome fragment by Real-time PCR
Detected DNA Virus	tested				Detected RNA Virus	not tested
	CMV	-	parvoB19	-		-/negative. +/positive. nt/not tested. (positive (+) does not immediately mean the production of infectious viral particles.)
	EBV	-	HBV	-
	HHV6	-	HTLV-1	-
	HHV7	-	HTLV-2	-
	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_3123

Reference
Pubmed id:8393236	A cell-type-specific transcription enhancer of type 16 human papillomavirus (HPV 16)-P97 promoter is defined with HPV-associated cellular events in human epithelial cell lines. Taniguchi A,Kikuchi K,Nagata K,Yasumoto S Virology. 1993 Aug;195(2):500-10
Pubmed id:1649895	Induction of chromosome abnormalities in mouse and human epidermal keratinocytes by the human papillomavirus type 16 E7 oncogene. Hashida T,Yasumoto S J Gen Virol. 1991 Jul;72 ( Pt 7)():1569-77
Pubmed id:1848315	Epidermal growth factor (EGF) elicits down-regulation of human papillomavirus type 16 (HPV-16) E6/E7 mRNA at the transcriptional level in an EGF-stimulated human keratinocyte cell line: functional role of EGF-responsive silencer in the HPV-16 long control region. Yasumoto S,Taniguchi A,Sohma K J Virol. 1991 Apr;65(4):2000-9
Pubmed id:2173586	Casein kinase II activities related to hyperphosphorylation of human papillomavirus type 16-E7 oncoprotein in epidermal keratinocytes. Hashida T,Yasumoto S Biochem Biophys Res Commun. 1990 Oct 30;172(2):958-64

Images

Cell No.	JCRB0141	Cell Name	PHK16-0b
LOT No.	10012010	Lot Specification	distribution
Medium	MCDB153 medium with 5 ug/ml insulin, 0.5 ug/ml hydrocortisone, 10 ug/ml transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 10 ng/ml EGF and 50 ug/ml BPE.	Temperature	37 C
Cell Density at Seeding	2.7 - 6.3 x 10^4 cells/ml	Methods for Passages	0.1% trypsin and 0.02% EDTA, Subculture every 1 week and change medium every 2-3 days.
Doubling Time	slow (approx. 1 week)	Cell Number in Vial (cells/1ml)	7.5 x 10^5
Viability at cell freezing (%)		Antibiotics Used	free
Passage Number	P28	PDL
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	0.1% methylcellurose, 10% DMSO in culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0141	Cell Name
LOT No.	08132009	Lot Specification	distribution
Medium	MCDB153, 5ug/ml Ins, 0.5ug/ml HC, 10ug/ml Tf, 0.1 mM PhosEtAmn & EtAmn, 10ng/ml EGF, 50ug/ml BPE.	Temperature	37 C
Cell Density at Seeding	2.5 - 7.1 x 10^4 cells/ml	Methods for Passages	0.1% trypsin and 0.02% EDTA, Subculture every 1 week and change medium every 2-3 days.
Doubling Time	81 hrs	Cell Number in Vial (cells/1ml)	1.0 x 10^6
Viability at cell freezing (%)	92.7	Antibiotics Used	free
Passage Number	P29	PDL
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	0.1% methylcellurose, 10% DMSO in culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0141	Cell Name
LOT No.	07192007	Lot Specification	distribution
Medium	MCDB153, 5ug/ml Ins, 0.5ug/ml HC, 10ug/ml Tf, 0.1 mM PhosEtAmn & EtAmn, 10ng/ml EGF, 50ug/ml BPE.	Temperature	37 C
Cell Density at Seeding	5-10 x 10^4 cells/ml	Methods for Passages	0.1% trypsin and 0.02% EDTA, Subculture every 1 week and change medium every 2-3 days.
Doubling Time	slow ( more than 4.5 days)	Cell Number in Vial (cells/1ml)	1.3 x 10^6
Viability at cell freezing (%)	89.5	Antibiotics Used	free
Passage Number	P29	PDL
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	0.1% methylcellurose, 10% DMSO in culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0141	Cell Name
LOT No.	09102003	Lot Specification	distribution
Medium	MCDB153, 5ug/ml Ins, 0.5ug/ml HC, 10ug/ml Tf, 0.1 mM PhosEtAmn & EtAmn, 10ng/ml EGF, 50ug/ml BPE.	Temperature	37 C
Cell Density at Seeding	ca. 1.5 x 10^3 cells/cm2	Methods for Passages	0.1% trypsin and 0.02% EDTA, Subculture every 1 week and change medium every 2-3 days.
Doubling Time	D.T. = 3.2 days	Cell Number in Vial (cells/1ml)	1.2 x 10^6
Viability at cell freezing (%)	76.2	Antibiotics Used	free (contains streptomycin in BPE used.)
Passage Number	P28	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	Confirmed as human by NP, G6PD (type B), AST, LD.
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	Yes	Exoteric Gene
Medium for Freezing	0.1% methylcellurose, 10% DMSO in culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0141	Cell Name	PHK16-0b
LOT No.	04182000	Lot Specification	distribution
Medium	MCDB153 medium supplemented with growth hormones. Ingredientd supplied by Yasumoto,S.	Temperature	37 C
Cell Density at Seeding	2 x 10^4 cells/sq.cm.	Methods for Passages	Cells are harvsted after treatment with 0.1% trypsin and 0.01% EDTA. Subculture once in 10-20 days and change medium once in 2-4 days.
Doubling Time	NT	Cell Number in Vial (cells/1ml)	1.7 x 10^6
Viability at cell freezing (%)	96.0	Antibiotics Used	free
Passage Number	P27	PDL	NT
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	G6PD(typeB), LD, and NP examined. Human confirmed.
Chromosome Mode	NT	Chromosome Information	NT
Surface Antigen	NT	DNA Profile (STR)	D5S818:9,11 D13S317:11 D7S820:9,11 D16S539:11,12 VWA:16 TH01:10 AM:XY TPOX:8 CSF1PO:12,13
Adhesion	Yes	Exoteric Gene	NT
Medium for Freezing	Mixture of the culture medium and FM-1 medium (1/1) with 10% DMSO.	CO2 Conc.	5 %
Viability immediately after thawing (%)		Additional information

Images

Cell No.	JCRB0141	Cell Name	PHK16-0b
LOT No.	02142000	Lot Specification	distribution
Medium	MCDB153 medium supplemented with growth hormones with no serum.	Temperature	37 C
Cell Density at Seeding	2.5 x 10^4 cells/sq.cm.	Methods for Passages	Cells harvested after treatment with 0.1% trypsin and 0.01% EDTA. Change medium every 2-3 days and subculture every 10-13 days.
Doubling Time	NT	Cell Number in Vial (cells/1ml)	1.6 x 10^6
Viability at cell freezing (%)	90.7	Antibiotics Used	free
Passage Number	P27	PDL	NT
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	G6PD(typeB), LD, NP, human confirmed.
Chromosome Mode	NT	Chromosome Information	NT
Surface Antigen	NT	DNA Profile (STR)	D5S818:9,11 D13S317:11 D7S820:9,11 D16S539:11,12 VWA:16 TH01:10 AM:XY TPOX:8 CSF1PO:12,13
Adhesion	Yes	Exoteric Gene	NT
Medium for Freezing	1/1 mixture of culture medium and FM-1 medium containing 10% DMSO.	CO2 Conc.	5 %
Viability immediately after thawing (%)		Additional information

Images

Cell No.	JCRB0141	Cell Name	PHK16-0b
LOT No.	12242015	Lot Specification	distribution
Medium	MCDB153 medium with 5 ug/ml insulin, 0.5 ug/ml hydrocortisone, 10 ug/ml transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 10 ng/ml EGF and 50 ug/ml BPE.	Temperature	37 C
Cell Density at Seeding	1.0 - 4.4 x 10^5 cells/mL	Methods for Passages	Cells are harvested with 0.1% trypsin and 0.02% EDTA. Medium change every 2 days and subculture every 10 days.
Doubling Time	approx. 5 days	Cell Number in Vial (cells/1ml)	2.4 x 10^6
Viability at cell freezing (%)	87.0	Antibiotics Used	free
Passage Number	P29	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	NT
Chromosome Mode	NT	Chromosome Information	NT
Surface Antigen	NT	DNA Profile (STR)
Adhesion	Yes	Exoteric Gene	NT
Medium for Freezing	10% DMSO, 0.1% methylcellurose in culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)	87.0	Additional information

Cell No.	JCRB0141	Cell Name	PHK16-0b
LOT No.	03282018	Lot Specification	distribution
Medium	MCDB153 medium with 5 ug/ml insulin, 0.5 ug/ml hydrocortisone, 10 ug/ml transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 10 ng/ml EGF and 50 ug/ml BPE.	Temperature	37 C
Cell Density at Seeding	0.5 - 1.5 x 10^5 cells/mL	Methods for Passages	Cells are harvested with 0.1% trypsin and 0.02% EDTA. The trypsin treatment should be terminated by adding soybean trypsin inhibitor (final conc, 0.2%). Medium change every 2 days and subculture every 10 days.
Doubling Time	approx. 4.5 days	Cell Number in Vial (cells/1ml)	1.7 x 10^6
Viability at cell freezing (%)	46.1	Antibiotics Used	free
Passage Number	P28	PDL
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, 0.1% methylcellurose in culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)	46.1	Additional information

Cell No.	JCRB0141	Cell Name	PHK16-0b
LOT No.	11242022	Lot Specification	distribution
Medium	KGM-Gold (Lonza Cat.# 00192060). Originally the instructed medium was MCDB153 medium with 5 ug/ml insulin, 0.5 ug/ml hydrocortisone, 10 ug/ml transferrin, 0.1 mM phosphorylethanolamine, 0.1 mM ethanolamine, 10 ng/ml EGF and 40-70 ug/ml BPE.	Temperature	37 C
Cell Density at Seeding	3 - 5 x 10^5 cells/mL	Methods for Passages	Cells were harvested after treatment with 0.1% trypsin and 0.02% EDTA. Trypsin action was terminated by 0.1% soybean trypsin inhibitor (Sigma, type IS).
Doubling Time	Slow	Cell Number in Vial (cells/1ml)	5.0 x 10^6
Viability at cell freezing (%)		Antibiotics Used	free
Passage Number	P29	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)	D5S818:9,11 D13S317:11 D7S820:9,11 D16S539:11,12 VWA:16 TH01:9.3 AM:X,Y TPOX:8 CSF1PO:12,13
Adhesion	Yes	Exoteric Gene
Medium for Freezing	10% DMSO, 0.1% methylcellurose in culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)	71.2	Additional information

JCRB Cell Bank

JCRB0141 PHK16-0b

Cell information

LOT Information