-Detailed Information [JCRB0156]-

JCRB0156 KHYG-1

Cell information

Important Notice(s)

[JCRB0156 KHYG-1: Tips of cultivation.]

The natural killar-like cell lines are generally weak for freeze-thawing and are sensitive to culture conditions. This is also true for KHYG-1 cell line.

(1). The cell density.

The growth of KHYG-1 tends to depend on high cell density. If the condition of cell line is not improved after thawing, the most simple method will be slant culture. The flask is slanted so as to achieve the local high cell density in the bottom of flask corner.

(2). Medium renewal method.

The human haematopoietic cell line is sensitive to culture conditions. The cell line itself secretes the growth factors and survival factors in the culture media. Therefore, excessive dilution or too frequent medium renewal may become rather harmful to cells. Simply add the medium to the old medium with cells.

(3). The pH of RPMI 1640

The media color indicated by phenol red should be orange. If the medium color is pink or purple, it is not suitable.

(4). Measures when growth is poor.

The increased concentration of fetal bovine serum (such as 20%) may improve the culture condition. Also, please try to increase the IL-2 concentration to 200 U/mL.

(5). About the unit of IL-2.

IL-2 products are available from many manufactureres. However, the activites/U(Unit)seem to be different between the products from different manufactures. We recommend to use the same manufacturer's IL-2 products described in "LOT Information - Medium" in JCRB website (in Cell Line Data Sheet shipped with cells). If you use IL-2 products from other othan Pepro Tech, we recommend to strat the culture with 200 U/mL.

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JCRB0156 KHYG-1細胞の培養について

[JCRB0156 KHYG-1細胞の培養について]
(The following sentence is in Japanese)

一般にヒト・ナチュラルキラー様細胞株は、凍結・解凍に弱く、培養条件に影響を受けやすい傾向があります。これはKHYG-1細胞株にも当てはまります。

(1) 細胞密度

　KHYG-1の増殖は高細胞密度に依存する傾向があります。細胞密度が低い状態ですと好調な増殖は期待できません。解凍時には過度の希釈を避けてください。

　お送りしているロットは、解凍培養時、10%血清、フラスコ平坦な状態で増殖することを確認しておりますが、解凍後、増殖が悪い場合には、局所的に細胞密度を上げるために、フラスコを傾けて角の部分に細胞を集めて培養すると効果があります。

(2) 細胞の継代、回収、培地交換

　細胞の継代は単純な希釈です。遠心の必要は特にありません。細胞にとって、お古の培地が好適な環境をもたらしますので、培地の全交換は避け、培地を追加、希釈する形で維持してください。

　本細胞はIL-2依存性に生存・増殖する細胞です。IL-2はその都度ストック溶液から添加するようにしています。培養の注意点はこのほか、次のような点がございます。

(3) RPMI1640のpH

　基礎培地のRPMI1640は、pHが高くなりやすい傾向があります。ヒト血球系細胞の場合、特に解凍培養時での高pHが細胞に大きな傷害を与える場合があります。培地がピンク色、紫色を帯びている場合、培養に不適です。この場合、希塩酸等で培地pHを適正に下げ、培地の呈色がオレンジ色になるようにしてください。

(4) 増殖が不良の際の措置

増殖が不調な場合には、増殖が好調になるまで血清濃度を20%濃度としてみてください。また、IL-2を200ユニット程度に増量してください。

(5) IL-2の単位について

IL-2は各社が販売していますが、同じ1ユニットでも活性が各社の製品間で異なるようです。JCRBウェブサイトのLot Information（細胞発送時に添付する細胞データシート）の培地情報に記したメーカーのIL-2を使用するようにしてください。また、Pepro Tech以外のIL-2を用いる場合は、200 U/mL IL-2を添加して培養を開始されることをお勧めいたします。




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CD25 expression in KHYG-1 cells

[CD25 expression in KHYG-1 cells]

There is difference in CD25 (low affinity IL-2R) expression between the original report of KHYG-1 and the FACS analysis by JCRB Cell Bank.

According to the original paper by the developer (Leukemia, 14: 922-930, 2000), the KHYG-1 is described as CD25 negative. However, JCRB's FACS analysis detected CD25 in KHYG-1.

[FOllowings are written in Japanese.]

KHYG-1細胞のCD25 (低親和性IL-2R)発現は、原著論文の記述とJCRBが実施したFACS解析で違いが認められました。

細胞樹立者らによる原著論文 (Leukemia, 14: 922-930, 2000)では、KHYG-1はCD25陰性と記述されています。しかし、JCRB細胞バンクのFACS解析ではCD25が検出されました。

KHYG-1細胞の使用に際しましては、原著論文の記述とは上記のような差異がありますことをご承知おきください。

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Cell type：general cells (View Pricing Information)

JCRB No.	JCRB0156	Cell Name	KHYG-1
Profile	Human natural killer cell line with a p53 point mutation as a model for p53-associated leukemogenesis and a model for differentiation of NK/T cells.	Other Name	KHYG
Animal	human	Strain
Genus	Homo	Species	sapiens
Sex	F	Age	45
Identity		Tissue for Primary Cancer	hemo-lymphocytic
Case history	aggressive NK cell leukemia	Metastasis	No
Tissue Metastasized		Genetics	p53 point mutation and cytogenetic characteristics
Life Span	infinite	Crisis PDL
Morphology	lymphocyte-like	Character	high NK/LAK activity, IFN gamma production etc.
Classify	tumor	Established by	Yagita,M.
Registered by	Yagita,M.	Regulation for Distribution	not defined
Comment		Year	2000
Medium	RPMI1640 medium containing with 10% fetal calf serum and 100 units/ml of rIL-2(Shionogi).	Methods for Passages	Simple dilution because of suspension cell culture. Subculture every 3-4 days with split ratio = 1/4.
Cell Number on Passage	Split 1/4 every 3-4 days.	Race	Jpanese
CO2 Conc.	5 %	Tissue Sampling	peripheral blood
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_2976

Reference
Pubmed id:10803526	A novel natural killer cell line (KHYG-1) from a patient with aggressive natural killer cell leukemia carrying a p53 point mutation. Yagita M,Huang CL,Umehara H,Matsuo Y,Tabata R,Miyake M,Konaka Y,Takatsuki K Leukemia. 2000 May;14(5):922-30

Images

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	11012010	Lot Specification	distribution
Medium	RPMI1640 medium with 10% heat-inactivated fetal bovine serum (FBS; GIBCO Cat.# 10099) and 100 units/ml recombinant human IL-2 (Pepro Tech; Cat. # 200-02)	Temperature	37 C
Cell Density at Seeding	approx. 5 x 10^5 cells/mL	Methods for Passages	Dilution. Avoid excessive dilution.
Doubling Time	approx. 54 hrs.	Cell Number in Vial (cells/1ml)	1.2 x 10^6
Viability at cell freezing (%)	88.1	Antibiotics Used	free
Passage Number	Unknown (9 at bank)	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	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - RPMI1640	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0156	Cell Name
LOT No.	07242006	Lot Specification	distribution
Medium	RPMI1640 medium with 10% fetal bovine serum and 100 units/ml IL-2 (corresponds to 50 ng/ml IL-2 Wako#097-03951) (FBS lot; GIBCO 4218333S)	Temperature	37 C
Cell Density at Seeding	2 - 5 x 10^5 cells/ml	Methods for Passages	dilution (avoid excessive dilution)
Doubling Time	approx. 30 hrs	Cell Number in Vial (cells/1ml)	1.6 x 10^6
Viability at cell freezing (%)	89	Antibiotics Used	free
Passage Number	Unknown (11 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	Confirmed as human by NP, G6PD, AST.
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0156	Cell Name
LOT No.	01212001	Lot Specification	distribution
Medium	RPMI1640 medium with 10% fetal bovine serum and 100 units/ml IL-2 (corresponds to 50 ng/ml IL-2 Wako#097-03951)	Temperature	37 C
Cell Density at Seeding	2 x 10^5 cells/ml	Methods for Passages	dilution (avoid excessive dilution)
Doubling Time		Cell Number in Vial (cells/1ml)	2.7 x 10^6
Viability at cell freezing (%)	80.9	Antibiotics Used	free
Passage Number	P11*	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	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS-base medium	CO2 Conc.	5%
Viability immediately after thawing (%)		Additional information

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	12092013	Lot Specification	distribution
Medium	RPMI1640 medium with 10% fetal bovine serum (GIBCO Cat. # 10091) and 100 units/mL recombinant human IL-2 (Pepro Tech; Cat. # 200-02)	Temperature	37 C
Cell Density at Seeding	1.5 -3.0 x 10^5 cells/mL	Methods for Passages	Simple dilution. (avoid excessive dilution)
Doubling Time		Cell Number in Vial (cells/1ml)	4.3 x 10^6
Viability at cell freezing (%)	96.4	Antibiotics Used	free
Passage Number	Unknown (9 at bank)	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	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - culture medium	CO2 Conc.	5%
Viability immediately after thawing (%)	96.4	Additional information

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	11202015	Lot Specification	distribution
Medium	RPMI1640 medium with 10% heat-inactivated fetal bovine serum (GIBCO Cat. # 10091) and 100 units/ml of recombinant human IL-2 (Pepro Tech; Cat. # 200-02)	Temperature	37 C
Cell Density at Seeding	3.7 - 5.7 x 10^5 cells/mL	Methods for Passages	Simple dilution (suspension culture). Avoid excessive dilution.
Doubling Time	approx. 30 hrs.	Cell Number in Vial (cells/1ml)	2.1 x 10^6
Viability at cell freezing (%)	87	Antibiotics Used	free
Passage Number	Unknown (9 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)	D5S818:9,11 D13S317:9,13 D7S820:10,11 D16S539:9 VWA:17 TH01:6 AM:X TPOX:9,11 CSF1PO:12
Adhesion	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - RPMI1640	CO2 Conc.	5%
Viability immediately after thawing (%)	87	Additional information

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	10182016	Lot Specification	distribution
Medium	RPMI1640 medium with 10% fetal bovine serum (FBS; Sigma Cat. # 172012) and 100 units/ml recombinant human IL-2 (Pepro Tech; Cat. # 200-02)	Temperature	37 C
Cell Density at Seeding	approx. 1 x 10^5 cells/mL	Methods for Passages	Simple dilution (suspension culture). Subculture every 3-4 days with split ratio = 1/4.
Doubling Time	approx. 25 hrs.	Cell Number in Vial (cells/1ml)	2.9 x 10^6
Viability at cell freezing (%)	85	Antibiotics Used	free
Passage Number	Unknown (9 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - RPMI1640	CO2 Conc.	5%
Viability immediately after thawing (%)	85	Additional information

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	10192018	Lot Specification	distribution
Medium	RPMI 1640 medium with 10% fetal bovine serum (FBS; Sigma Cat. # 172012) and 100 units/mL recombinant human IL-2 (Pepro Tech; Cat. # 200-02)	Temperature	37 C
Cell Density at Seeding	approx. 1 x 10^5 cells/mL	Methods for Passages	Simple dilution (suspension culture). Subculture every 3-4 days with split ratio = 1/4.
Doubling Time	approx. 27 hrs.	Cell Number in Vial (cells/1ml)	9.6 x 10^5
Viability at cell freezing (%)		Antibiotics Used	free
Passage Number	Unknown (9 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - RPMI 1640	CO2 Conc.	5%
Viability immediately after thawing (%)	87.3	Additional information

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	12072020	Lot Specification	distribution
Medium	RPMI1640 medium(GIBCO：Cat No.;11875-093) with 10% heat inactivated fetal bovine serum(SIGMA,Cat#172012,Lot#12J396) and IL-2(100 U/ml) (Pepro Tech,recombinant human IL-2)	Temperature	37 C
Cell Density at Seeding	0.38-1.0x10^5 cells/ml	Methods for Passages	dilution
Doubling Time	NT	Cell Number in Vial (cells/1ml)	1.5x10^6
Viability at cell freezing (%)	89.10	Antibiotics Used	free
Passage Number	p10*	PDL	NT
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis	NT
Chromosome Mode	NT	Chromosome Information	NT
Surface Antigen	NT	DNA Profile (STR)	D5S818:9,11 D13S317:9,13 D7S820:10,11 D16S539:9 VWA:17 TH01:6 AM:X TPOX:9,11 CSF1PO:12
Adhesion	No	Exoteric Gene	NT
Medium for Freezing	BAMBANKER(LYMPHOTEC Inc.,CS-02-001,NIPPON Genetics Co., LTD)	CO2 Conc.	5%
Viability immediately after thawing (%)	72.70	Additional information

Images

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	12212020	Lot Specification	distribution
Medium	RPMI 1640 medium with 10% fetal bovine serum (FBS; Sigma Cat. # 172012) and 100 units/mL recombinant human IL-2 (Pepro Tech; Cat. # 200-02)	Temperature	37 C
Cell Density at Seeding	1 - 3 x 10^5 cells/mL	Methods for Passages	Simple dilution (suspension culture). Subculture every 3-4 days with split ratio = 1/4.
Doubling Time	approx. 40 hrs.	Cell Number in Vial (cells/1ml)	3.8 x 10^6
Viability at cell freezing (%)		Antibiotics Used	free
Passage Number	Unknown (18 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - RPMI1640	CO2 Conc.	5%
Viability immediately after thawing (%)	79	Additional information

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	07292022	Lot Specification	distribution
Medium	RPMI 1640 medium with 10% fetal bovine serum (FBS; GIBCO Cat. # 10437) and 100 units/mL recombinant human IL-2 (Pepro Tech; Cat. # 200-02). Use 20% FBS at the intiation of culture.	Temperature	37 C
Cell Density at Seeding	3 - 5 x 10^5 cells/mL	Methods for Passages	Simple dilution (suspension culture)
Doubling Time		Cell Number in Vial (cells/1ml)	6.6 x 10^5
Viability at cell freezing (%)		Antibiotics Used	free
Passage Number	Unknown (12 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)	D5S818:9,11 D13S317:9,13 D7S820:10,11 D16S539:9 VWA:17 TH01:6 AM:X TPOX:9,11 CSF1PO:12
Adhesion	No	Exoteric Gene
Medium for Freezing	5% DMSO, 20% FBS - RPMI1640	CO2 Conc.	5%
Viability immediately after thawing (%)	48.2	Additional information	Many dead cells will appear immediately after thawing, but the growth and recovery after thawing was confirmed.

Cell No.	JCRB0156	Cell Name	KHYG-1
LOT No.	05222023	Lot Specification	distribution
Medium	RPMI 1640 medium with 10% fetal bovine serum (FBS; GIBCO Cat. # 10437) and 100 units/mL recombinant human IL-2 (Pepro Tech; Cat. # 200-02). Use 20% FBS at the intiation of culture.	Temperature	37 C
Cell Density at Seeding	1.2 - 2.4 x 10^5 cells/mL	Methods for Passages	Simple dilution (suspension culture)
Doubling Time	approx. 33 hrs.	Cell Number in Vial (cells/1ml)	4.1 x 10^6
Viability at cell freezing (%)	83	Antibiotics Used	free
Passage Number	Unknown (10 at bank)	PDL
Sterility: MYCOPLASMA	-	Sterility: BACTERIA	-
Sterility: FUNGI	-	Isozyme Analysis
Chromosome Mode		Chromosome Information
Surface Antigen		DNA Profile (STR)
Adhesion	No	Exoteric Gene
Medium for Freezing	10% DMSO, 20% FBS - RPMI1640	CO2 Conc.	5％
Viability immediately after thawing (%)	78	Additional information