Table 1.

Human Primary Cholangiocarcinoma Cell Lines Established between 1971 and 2000

Cell no.	Year	Country	Cell name	Diagnosis	Isolation source	Digestion method	Sorting method	Maintenance medium	Cell morphology	Doubling time	References
1	1971	USA	RPMI 7451	CCA	—	—	—	Eagle's MEM + 10% FBS + nonessential amino acids + 60 pg/mL gentamicin	Tightly adherent, monolayer, polygonal shaped cells	—	17,39,40
2	1975	Japan	H-1 or H1	CCA	—	—	—	—	—	—	40,41
3	1981	Japan	OZ	IHCCA	Ascites	No digestion	Colony isolation by trypsin and EDTA-soaked filter; other cells removed by enzymatic and mechanical treatments	Williams' E + 10% newborn calf serum + P/S	Large nucleus with 1 to 2 nucleoli; dark cytoplasm; high nucleus/cytoplasm ratio; pavement-like proliferation. Abundant production of gel-like substance	48 hours	42
4	1984	Germany	EGI-1	CCA	Primary tumor	—	—	MEM + 10% FBS + 2× MEM amino acids (essential and nonessential) + 4 mmol/L l-glutamine + 1 mmol/L sodium pyruvate	Monolayer, adherent, polymorphic cells	45–50 hours	43
5	1985	Japan	HChol-Y1	CCA	Primary tumor	No digestion	Fibroblasts spontaneously disappeared in 2 months	Ham's F12	Uniform, monolayer cells; including abundant granules; prominent round nucleus with multiple nucleoli	52 hours	44
6	1985	Germany	SK-ChA-1 or WITT	EHCCA	Ascites	No digestion	Light trypsin treatment	CMRL + 15% + P/S	Adherent, spindle- to polygonal shaped, polymorphic cells; proliferating as single adherent cells or small clusters	48 hours	45,46
7	1987	Japan	KMCH-1	IHCCA + HCC	Primary tumor	0.5 U/mg type IV collagenase in PBS, 37°C, 30 minutes	Colony isolation by trypsin	DMEM + 20% FBS + 35 μmol/L sodium bicarbonate + P/S	Large, round nucleus with multiple nucleoli; abundant clear cytoplasm; pavement-like proliferation; microvilli on the luminal surfaces	39 hours	47
8	1988	Japan	HuH-28	IHCCA	Primary tumor (frozen-thawed)	500 U/mL trypsin	—	RPMI 1640 + 20% FBS + %0.2 lactalbumin hydrolysate	Spindle- to polygonal shaped cells	80 hours	48
9	1989	Japan	HuCC-T1	IHCCA	Ascites	No digestion	IS-RPMI medium used to eliminate fibroblasts	RPMI 1640 + 0.2% lactalbumin hydrolysate	Polygonal to spindle-shaped, abundant and clear cytoplasm, proliferation in pavement arrangement	74 hours	49,50
10	1989	Japan	MEC	CCA	Pleural effusion	No digestion	—	RPMI 1640 + 20% FBS	Polymorphic, epithelial-like cells; high nucleus/cytoplasm ratio	40 hours	51
11	1990	USA	PCI:SG231	IHCCA	Primary tumor	Type II collagenase, 37°C, 3 hours	—	α MEM (Earle's salts) with nucleosides + 10% FBS + 60 pg/mL gentamicin	Tightly adherent, monolayer, polygonal shaped cells; nonadherent cell clusters that formed three-dimensional tubular structures resembling bile ducts	—	40
12	1991	Thailand	HuCCA-1	IHCCA	Primary tumor	No digestion	Fibroblasts gradually decreased with each passage and disappeared in a month	Ham's F12 + P/S	Monolayer, adherent, polygonal shaped epithelial cells with occasionally multiple, round to oval nuclei; granule-filled cytoplasms; piling up of cells and occasional gland-like appearances	55 hours	52
13	1991	Japan	KMBC	EHCCA	Xenograft	Collagenase 150–250 U/mg, 37°C, 90–110 minutes	Fibroblasts scraped away and completely disappeared after several passage	DMEM + 5% FBS + 12 mmol/L sodium bicarbonate + P/S	Polymorphic epithelial-like cells; one or more large, irregular, round to oval nuclei with a few prominent nucleoli; relatively poor, round to polygonal cytoplasm; pavement-like proliferation; tubular formation	30 hours	53,54
14	1992	USA	CC-LP-1	IHCCA	Primary tumor	0.05% (w/v) collagenase type IV, 0.002% (w/v) DNase type 1, 90 minutes	Centrifugation on double-layer (75%/100% v/v) Ficoll-Hypaque gradients; fibroblasts removed by differential trypsinization	DMEM + 15% FBS + 2 mmol/L l-glutamine + antibiotics	Cobblestone-like proliferation of monolayers; stratification of cells in some areas	180 hours	55
15	1992		CC-SW-1							72 hours
16	1992	Japan	KMC-1	IHCCA	Xenograft	0.5 mg/nL type IV collagenase in PBS, 37°C, 60–80 minutes	Tumor cells suppressed the fibroblasts by the time	DMEM + 10% or 5% FBS	Monolayer, pavement-like proliferation; clear cytoplasm, oval-shaped nuclei; tubular formation; some cells with mucin in the cytoplasm	54 hours	56
17	1993	China	QBC939	EHCCA	Primary tumor	Type I collagenase, DNase I, plasminase	Fibroblasts gradually decreased by passaging	RPMI 1640 + 10% FBS	Monolayer, polymorphic, adherent cells; round or oval nuclei; high nucleus/cytoplasm ratio	24 hours	57,58
18	1994	Japan	TK	EHCCA	Ascites	No digestion	Colony isolation using 0.33% agar	RPMI 1640 + 15% FBS + 2 mmol/L glutamine + 1 mmol/L sodium pyruvate	Monolayer, adherent proliferation; forming gland-like structures; lobated or dark, large nuclei	29 hours	59
19	1995	Japan	OCUCh-LM1	EHCCA	Primary tumor	No digestion	Fibroblasts gradually decreased and disappeared in 1 month	DMEM + 10% FBS + 2 mmol/L l-glutamine + 0.5 mmol/L sodium pyruvate + P/S	Monolayer, pavement-like proliferation; clear cytoplasm and oval nuclei	31 hours	60
20	1995	Japan	TFK-1	EHCCA	Primary tumor	1000 U/mL dispase, 37°C, 30 minutes	Fibroblasts removed by mechanical scraping and differential attachment selection with trypsin	RPMI 1640 + 10% FBS + P/S	Polygonal epithelial monolayers; pavement-like proliferation	37 hours	61
21	1996	Japan	KMCH-2	IHCCA + HCC	Primary tumor	150–250 U/mg collagenase in PBS, 37°C, 90–110 minutes	Fibroblasts removed by mechanical scraping	DMEM + 5% FBS + 12 mmol/L sodium bicarbonate + P/S	Monomorphic polygonal cells; large round to oval nuclei with a few prominent nucleoli; pavement-like proliferation	44 hours at 20th passage; 32 hours at 55th passage	62
22	1997	Japan	ETK-1	IHCCA	Ascites	No digestion	Limiting dilution	RPMI 1640 + 10% FBS + 10 mmol/L HEPES + 2 mmol/L l-glutamine + 0.1 mmol/L nonessential amino acids + 1 mmol/L sodium pyruvate + 0.005 mmol/L β-mercaptoethanol	Small polygonal cells; round to oval nuclei and prominent nucleoli; uniform, monolayer with a pavement-like proliferation	71 hours	41
23	1997	Japan	ICBD-1	EHCCA	Primary tumor	No digestion	Fibroblasts gradually decreased and disappeared in 1 month	RPMI 1640 + 10% FBS + 0.5 mmol/L sodium bicarbonate + 2 mmol/L l-glutamine + P/S	Large round to oval nuclei with a few nucleoli; relatively poor, polygonal to oval cytoplasm; pavement-like proliferation	31.5 hours	63
24	1997	Japan	SSP-25	IHCCA	Primary tumor	No digestion	Fibroblasts gradually disappeared, limiting dilution	RPMI 1640 + 10% FBS	Spindle-shaped cells	64 hours	64
25			RBE						Monolayer of polygonal cells, pavement-like proliferation	45 hours
26	1998	Japan	TBCN-1	EHCCA	Primary tumor	—	—	RPMI 1640 + 10% FBS	—	54 hours	65
27	1999	Thailand	KKU-213A	IHCCA	Primary tumor	0.25% trypsin-EDTA, 37°C, 1 hour	—	DMEM + 10% FBS + A/A	Small, spindle-shaped cells	23 hours	26
28			KKU-213B						Irregular polygonal cells; high nucleus to cytoplasmic ratio; patch-like structure	24.5 hours
29			KKU-213C						Irregular polygonal cells; high nucleus to cytoplasmic ratio	25.6 hours
30	1999	Japan	YSCCC	CCA	—	—	—	RPMI 1640 + 10% FBS	Lymphocyte-like	—	66, https://cellbank.brc.riken.jp/cell_bank/CellInfo/?cellNo=RCB1549&lang=, last accessed May 5, 2022
31	2000	Japan	HBDC	EHCCA	Ascites	No digestion	Centrifugation on triple-layer (75%/100%/25) Ficoll-Hypaque gradients; colony isolation using porcelain cloning rings	Williams' E + 10% FBS + 2 ng/mL HGF + 2 mmol/L l-glutamine + 6 mmol/L glucose + 0.5 mmol/L sodium bicarbonate + 100 ng/mL kanamycin + 10 ng/mL fungizone	Polygonal or spindle-shaped polymorphic cells; occasional large vacuoles in the cytoplasm; forming small clusters or clumps; one or more large, irregular, round or oval nuclei with a few prominent nucleoli; pavement-like proliferation	32 hours	67

A/A, antibiotic + antimycotic; CCA, cholangiocarcinoma; CMRL, Connaught Medical Research Laboratories medium; DMEM, Dulbecco's modified Eagle's medium; EHCCA, extrahepatic cholangiocarcinoma; FBS, fetal bovine serum; HCC, hepatocellular carcinoma; IHCCA, intrahepatic cholangiocarcinoma; MEM, minimal essential medium; P/S, penicillin/streptomycin.