Development and Characterization of Human Primary Cholangiocarcinoma Cell Lines (original) (raw)

Abstract

Cholangiocarcinoma (CCA) is the second most common primary liver tumor and is associated with late diagnosis, limited treatment options, and a 5-year survival rate of around 30%. CCA cell lines were first established in 1971, and since then, only 70 to 80 CCA cell lines have been established. These cell lines have been essential in basic and translational research to understand and identify novel mechanistic pathways, biomarkers, and disease-specific genes. Each CCA cell line has unique characteristics, reflecting a specific genotype, sex-related properties, and patient-related signatures, making them scientifically and commercially valuable. CCA cell lines are crucial in the use of novel technologies, such as three-dimensional organoid models, which help to model the tumor microenvironment and cell-to-cell crosstalk between tumor-neighboring cells. This review highlights crucial information on CCA cell lines, including: i) type of CCA (eg, intra- or extrahepatic), ii) isolation source (eg, primary tumor or xenograft), iii) chemical digestion method (eg, trypsin or collagenase), iv) cell-sorting method (colony isolation or removal of fibroblasts), v) maintenance-medium choice (eg, RPMI or Dulbecco's modified Eagle's medium), vi) cell morphology (eg, spindle or polygonal shape), and vii) doubling time of cells.

Cholangiocarcinoma (CCA) is a heterogeneous group of malignancies originating from the biliary tree.1 CCA accounts for approximately 3% of all malignancies of the gastrointestinal system and is the second most common primary hepatic tumor, after hepatocellular carcinoma.2,3 The prevalence of CCA varies broadly by region; while the prevalence is 1.6 per 100,000 people in the United States, it could be as high as 85 to 90 per 100,000 people in northeastern Thailand, where infection with Opisthorchis viverrini, a CCA risk factor, is endemic.4 Several additional risk factors for cholangiocarcinogenesis have been identified, such as Clonorchis sinensis infection, bile duct cyst, primary sclerosing cholangitis, hepatolithiasis, cholelithiasis, and inflammatory bowel disease.5 Despite the development of state-of-the-art therapies, the 5-year survival rate of CCA is still under 30%.6,7

CCAs are commonly classified, according to anatomic location along the biliary tree, as extrahepatic or intrahepatic (EHCCA or IHCCA).8 EHCCAs are subcategorized as perihilar or distal.8 CCAs can also be subclassified based on macroscopic growth pattern (mass forming, periductal infiltrating, intraductal, or mixed), cell of origin (cholangiocytes, goblet cells, hepatic stem cells, or biliary tree stem/progenitor cells), and/or microscopic features (adeno, squamous, adenosquamous, mucinous, undifferentiated, or sarcomatous).9,10

Although the importance of organ-specific or cancer-specific microenvironment has been recently highlighted due to cell-to-cell crosstalk, two-dimensional cancer cell lines are one of the best sources for investigating the respective cancer type.11,12 CCA cell lines have been used for almost 50 years to: i) better understand CCA properties, ii) investigate treatment options, iii) model the disease in vitro, and iv) generate in vivo xenograft models.13, 14, 15, 16 RPMI 7451 is the first known CCA cell line isolated by George Eugene Moore and his team.17 Dr. Moore, an oncologist, surgeon, accomplished scientist, and director of the RPMI, pioneered many cancer studies, established several additional cancer cell lines, and formulated RPMI 1640, a widely used cell-growth medium.18, 19, 20, 21, 22

There is currently a growing interest in CCA-related basic and translational research, due to poor outcomes with the currently available treatment options. For recently developed novel experimental models [eg, three-dimensional (3D) organoids] and treatments, CCA cell lines have been used for the identification of novel mechanistic pathways, biomarkers, and disease-specific genes. Therefore, previously isolated human primary CCA cell lines, their isolation methods, and known essential characteristics are summarized herein to help researchers in their future studies.

Human Primary CCA Cell Lines

The first CCA cell line was established about a half-century ago, in 1971 in the United States17; more than half of currently available CCA cell lines were generated in Japan between 1975 and 1990. To date, researchers from Italy, Germany, China, South Korea, and Thailand have reported on the isolation of CCA cell lines and related studies. In the past 2 decades, researchers have reported the isolation of multiple CCA cell lines in Thailand more than in any other country, possibly due to the high prevalence of the disease in that geographic region.4

Each CCA cell line is unique in reflecting a specific genotype, sex-related properties, and patient-related signatures, making them especially valuable for scientific and commercial applications. It would undoubtedly be helpful to have an international and collective cell bank that includes cell-identification analysis from each study to appropriately define the CCA properties and prevent any misidentification of the existing cell lines.23 For instance, the ETK-1 CCA cell line was retracted in 2004 after a short tandem repeat polymorphism analysis revealed that the ETK-1 and SSP-25 cell lines were identical.24,25 However, later, it was understood that SSP-25 should have been retracted instead of ETK-1 because the SSP-25 and RBE cell lines were isolated from the same patient, but their short tandem repeat profiling results did not match (https://cell.brc.riken.jp/en/rcb/rbessp-25_announce, last accessed May 5, 2022). As a result, the ETK-1 cells have been distributed under the name of SSP-25 since 2004, but it is unclear when that cell line was initially misidentified. It is also unclear what happened to the SSP-25 cell line. Similarly, the M156, M213, and M214 cell lines were believed to have been isolated from three separate patients; however, results from recent short tandem repeat profiling showed that they were isolated from the same patient.26

Cross-contamination of cell lines is a long-standing problem that has resulted in scientific errors27 and may initiate inaccurate data chains. Various levels of the cell-culturing process have been blamed for cross-contamination, including: i) accidental inoculation, ii) mislabeling, iii) confusion in freeze-thawing, iv) working with more than one cell line in a biosafety cabinet at the same time, and v) contamination of the stock bottle of media with cells.28,29 Additionally, cancer cells may carry a greater risk for cross-contamination due to their greater capacity for proliferation. Even a minimal amount of inoculation of cancer cell lines could suppress other cell lines in the culture and take their place over time. Therefore, the preservation and maintenance of the cell line is as important and challenging as is the establishment of one.

In a different case, CHGS was misidentified as a CCA cell line in 2015 by Zach et al30 and listed in the Cellosaurus database (accession number CVCL_M272; https://web.expasy.org/cellosaurus, last accessed May 5, 2022). However, the first mention of CHGS, in 1988 by Katoh et al,31 indicates that CHGS is a CCA tissue line passaged in mice, and that cancer cells have not been isolated from this tissue line. Similarly, the CC-CL-1 cell line was studied as a CCA cell line in a research study; however, none of the references cited in the study contain information regarding the CC-CL-1 cell line, generating doubts about its credibility.32

In addition to primary CCA cell lines, derivative CCA cell lines have been established for drug resistance studies. QBC939/ADM is doxorubicin resistant; HuCCT1-G100, YSCCC-G100, RTFK-1, KKU-M139/GEM, KKU-213B/GEM, MT-CHC01R1.5, and SNU-1196/GR are gemcitabine resistant; and KKU-M055/46 and KKU-213B/246 are 5-fluorouracil–resistant CCA cell lines.16,33, 34, 35, 36, 37 The KKU-213L5 cell line is a derivative of the KKU-213A cell line, with high metastatic activity.38

Tables 1 and 2 summarize the CCA cell lines available between 1971 and 2000, and between 2001 and 2021, respectively, from the English and non-English published literature. Tables 1 and 2 include: i) the type of CCA (eg, intra- or extrahepatic), ii) isolation source (eg, primary tumor or xenograft), iii) tissue-digestion method (eg, trypsin or collagenase), iv) cell-sorting and -purification methods (colony isolation or removal of fibroblasts), v) maintenance-medium choice (eg, RPMI or DMEM), vi) cell morphology (eg, spindle or polygonal shape), and vii) doubling time of cells.

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

Table 2.

Human Primary Cholangiocarcinoma Cell Lines Established between 2001 and 2021

Cell no. Year Country Cell name Diagnosis Isolation source Digestion method Sorting method Maintenance medium Cell morphology Doubling time References
1 2001 Republic of Korea Choi-CK IHCCA Primary tumor No digestion Differential trypsinization, scraping, or G418 treatment (100 μg/mL) Opti-MEM + 10% FBS + 30 mmol/L sodium bicarbonate + antibiotics Polygonal and compact cells with indistinct cell boundaries — 75
2 Cho-CK Polygonal and compact cells with indistinct cell boundaries
3 JCK Polygonal morphology and some spindle-shaped cells
4 SCK Monolayer, epithelioid or spindle-shaped cells; fewer polygonal shaped cells
5 2002 Thailand HubCCA-1 IHCCA Intrahepatic biliary fluid — — Ham's F12 + 20% FBS + P/S Adherent, monolayer, polygonal to spindle-shaped cells — 76,77
6 2002 Thailand KKU-100 IHCCA Primary tumor 0.025% trypsin-EDTA, 37°C, 1 hour Fibroblasts removed with a cell scraper and by differential trypsinization Ham's F12 + 20% FBS + P/S Compact, polygonal to spindle-shaped cells; floating or clumping in a confluent monolayer; large nucleus containing two to five nucleoli and a clear cytoplasm 72 hours 77,78
7 2002 Republic of Korea SNU-245 EHCCA Primary tumor No digestion Differential trypsinization used to remove fibroblasts RPMI 1640 + 10% FBS Monolayer, adherent cells; trabecular arrangement with acinar formation 54 hours 79
8 SNU-1079 IHCCA Monolayer, adherent cells; pleomorphic appearance with multiple cytoplasmic processes; numerous cytoplasmic vacuoles in some cells; some multinucleated cells 72 hours
9 SNU-1196 EHCCA Monolayer, adherent cells; trabecular pattern consisted of spindle to polygonal shaped cells having vesicular nuclei and multiple small nucleoli 48 hours
10 2004 Japan TKKK IHCCA — — — DMEM (low glucose) + 10% FBS Adherent, monolayer, epithelial-like cells; pavement-like proliferation — 80
11 2005 Thailand KKU-M055 IHCCA — — — Ham's F-12 + 10% FBS + P/S — — 81
12 KKU-OCA17
13 2005 Japan TBCN6 EHCCA Xenograft 0.25% trypsin in PBS, 37°C, 10 minutes — DMEM + 10% FBS Adherent, monolayer, polygonal cells 38 hours 82
14 TGBC-47
15 2006 Thailand KKU-M139 CCA Primary tumor — — Ham's F12 or RPMI 1640 + 10% FBS + P/S Adherent, monolayer, polygonal cells; pavement-like proliferation 17 hours 16,76
16 2006 Thailand RMCCA-1 IHCCA Primary tumor No digestion Differential trypsinization used to remove fibroblasts Ham's F12 + 20% FBS + EGF + 250 μg/mL amphotericin + P/S Circular to spindle shape with many processes and ornamental fringes; granulated nucleus and cytoplasm 48 hours 83
17 2007 China HKGZ-CC IHCCA Primary tumor 1200–2000 U/mL collagenase and DNase in 10 mL/g DPBS — DMEM Adherent, monolayer, epithelial-like cells 48 hours 84
18 2007 Japan IHGGK IHCCA — — — RPMI 1640 + 10% FBS + P/S Adherent, monolayer, epithelial-like cells 85, http://www2.idac.tohoku.ac.jp/dep/ccr/TKGdate/TKGvo106/0623.html, last accessed May 5, 2022
19 2009 Thailand CL-2 CCA — — — DMEM + 15% FBS + P/S — — 86,87
20 CL-6
21 CL-19
22 2010 Japan NCC-BD1 EHCCA Xenograft No digestion Fibroblasts removed by mechanical scraping RPMI 1640 + 10% FBS + 2 mmol/L l-glutamine + P/S Adherent, monolayer, epithelial-like cells; pavement-like proliferation — 88
23 NCC-BD2 EHCCA
24 NCC-CC1 IHCCA
25 NCC-CC3-1 IHCCA
26 NCC-CC3-2 IHCCA
27 NCC-CC4-1 IHCCA
28 2013 China HCCC-9810 IHCCA — — — RPMI 1640 + 10% FBS + P/S — 20 hours 89
29 2015 Italy MT-CHC01 IHCCA Xenograft 200 U/mL collagenase, 3 hours, 37°C — Knockout/DMEM/F-12 + 10% FBS + P/S Monolayer, adherent cells 40 hours 90
30 2016 USA ICC1 IHCCA — Trypsin, 30 minutes, 37°C — RPMI or DMEM/F12 + 5% FBS + P/S — 91
31 ICC2
32 ICC5
33 2016 China ZJU-0826 EHCCA Primary tumor — Fibroblasts eliminated by differential trypsinization and differential attachment RPMI 1640 + 10% FBS + antibiotics Monolayer, adherent, homogeneous cells with characteristic loose pleomorphic cells and rare multinucleated cells 63 hours 92
34 ZJU-1125 IHCCA Monolayer, epithelial-like, adherent cells; polygonal, occasionally multinucleated 44 hours
35 2017 Germany CCC-5 EHCCA Pleural effusion No digestion — DMEM + 20% FBS: KFSM - 2:1 Monolayer, spindle- to polygonal shaped cells, pavement-like proliferation; anaplastic, multinucleated giant cells 60 hours 93
36 2017 Thailand KKU-023 IHCCA Primary tumor 1 mg/mL collagenase, 37°C, 30–45 minutes Fibroblasts aseptically removed with a cell scraper Ham's F12 + 10% FBS + P/S + nonessential amino acids + 12.5 mmol/L HEPES + 50 μg/mL cefazolin + 10 μg/mL ciprofloxacin + 2.5 μg/mL amphotericin B + 5 μmol/L ROCK inhibitor Ovoid to cuboid shape polygonal cells; seldom multinucleated; forming compact monolayer with occasional multinucleated cells 34 hours 94
37 KKU-452 EHCCA Spindle-shaped cells; seldom multinucleated; segregated and spread surround 17 hours
38 2018 China ICC-1 IHCCA Primary tumor No digestion — — — — 73
39 ICC-2
40 2019 Thailand KKK-D049 IHCCA Xenograft 1000 U/mL collagenase + 0.1 mg/mL DNase I, 3 hours Stromal cells sequentially removed by partial trypsinization and mechanical removal DMEM + 10% FBS + P/S Epithelial-like cells; high nuclear to cytoplasmic ratio; tight clustering — 95
41 KKK-D068 Epithelial-like, polygonal and spindle-shaped cells; high nuclear to cytoplasmic ratio —
42 KKK-D131 Epithelial-like, polygonal and spindle-shaped cells; high nuclear to cytoplasmic ratio —
43 KKK-D138 Epithelial-like, polygonal and spindle-shaped cells; high nucleus-to-cytoplasm ratio —
44 2021 Italy 82.3 IHCCA Xenograft Collagenase 200 U/mL, 3 hours, 37°C 10% FBS + P/S + gemcitabine Monolayer, adherent, epithelial-like cells 53 hours 74

Overview of Isolation Methods

The general principles of the CCA cell–isolation protocol are summarized in Figure 1. CCA cell isolation requires either a solid sample (eg, primary tumor or xenograft) or a liquid sample (eg, ascites or pleural effusion). Samples can come from autopsy, surgery, paracentesis, or animal harvesting. Samples can be transferred into Ham's F12 culture medium at 4°C.52 It is better to process the samples as soon as possible to avoid ischemia-related problems in cells. If the CCA sample needs to be preserved, it can be freeze-thawed in 10% dimethyl sulfoxide in culture medium48 or can be preserved in histidine-tryptophan-ketoglutarate organ-preservation solution at 4°C until tissue processing.68

Figure 1.

Figure 1

CCA cell isolation method. CCA cell lines can be isolated from either fresh samples (eg, HuCCA-1 cell line) or frozen-thawed samples (eg, huh-28 cell line) from primary tumors or from xenografts, kept in different mediums, after manual dissociation, plated with or without digestion.

CCA cell lines can be isolated using several methods.44,47 Herein, the general perspective of isolation protocols is briefly summarized. All tissue processing should be performed under aseptic conditions in a biological safety cabinet with sterile or disposable surgical instruments. The first and the most essential step of CCA cell isolation from solid samples is cutting the tissue into small pieces (>1 mm in diameter).44, 45, 46, 47 After that, the protocol can be continued either with or without enzymatic digestion. For the protocols without enzymatic digestion, a stainless-steel mesh is helpful for releasing CCA cells.44 Chemical digestion usually proceeds at 37°C, and the digestion time is dependent on the concentration and type of the enzyme used (Table 1). Several types of collagenases (types I, II, and IV) and trypsin have been successfully used for chemical digestion (Table 1). Plasminase to digest fibrin clumps and DNase I to lyse DNA leaked into cell suspension can be included in the dissociation protocol (Figure 1).57

After chemical dissociation, the cell suspension should be filtered with 70 μm of mesh and centrifuged47; the pellet can then be resuspended in a variety of media conditions [RPMI, Dulbecco's modified Eagle medium, minimum essential medium, William's, and Ham's F12].42, 43, 44,47,49 Stromal fibroblasts are the primary contaminating cells in CCA cell cultures.69 After cell attachment, CCA cells can be purified using scraping of the contaminating cells, colony isolation, and/or passaging cells until all contaminating cells have been removed (Figure 1).42,44,49

Need for More CCA Cell Lines

Cell lines provide indispensable in vitro model systems for the assessment of features of cancer cells, since they are direct descendants of the primary tumors.12 Under the right conditions, most of the phenotypic and genotypic properties of the primary tumor are preserved in cancer cell lines.11 After >50 years of research, these features have allowed for the establishment of thousands of cell lines from various malignancies, of which only 70 to 80 belong to the CCA family (Tables 1 and 2). While each new cell line is not necessarily superior to previous lines, collectively, they have been important for the understanding of common and differing features of CCA, including: i) antigenic variations, ii) new genotypes, and iii) enzymes that sustain tumorigenic growth to drug sensitivities or resistances.

One reason that CCA remains difficult to cure is the lack of a precise understanding of oncogenesis.70 The established CCA cell lines can be utilized to identify the properties of cells that can help in predicting positive or negative responses to antigen- or pathway-targeted therapies. CCA cell lines could be powerful in determining many aspects of CCA phenotypes, especially when used in a 3D microenvironment in the presence of other liver cells to study the impact of adjacent cells in the tumor milieu.71 The 3D tumor organoid system can be used for the identification of candidate novel therapies for future clinical trials.72

In a recent study, an established IHCCA cell line was used for understanding the impact of RA190, a proteasome subunit ADRM1 inhibitor that induces cell apoptosis, in a pathway-targeted therapy model.73 Although the outcomes of that in vitro study were encouraging, the study did not evaluate the roles of the tumor microenvironment and other CCA cell lines to determine whether a genotypic difference within the CCA lines may have modulated (increased or decreased) the response and sensitivity to RA190.73 A counterexample is evidenced by the recent discovery in Italy of a novel IHCCA cell line characterized by the presence of genes encoding resistance to fluorouracil, carboplatin, and oxaliplatin—drugs actively and frequently used in CCA treatment.74

Future Perspectives

The establishment of new, well-defined, and well-characterized CCA cell lines is crucial in enhancing the understanding of this aggressive disease. The establishment of oncogenesis through the cancer cells provides information on their drug-sensitivity and drug-resistance mechanism, and helps to identify possible novel drug targets. The inclusion of tumor stromal cells, such as hepatic stellate cells, liver sinusoidal endothelial cells, and tumor-associated macrophages, in the CCA cell lines makes these culture models more representative of the CCA tumor microenvironment (ie, 3D tumor organoids)71 (Figure 2). Therefore, the inclusion of multiple other cell types will help to determine the ways in which CCA cells interact with other neighboring cells and alter their genotypes, phenotypes, and transcriptomic profiles.

Figure 2.

Figure 2

CCA organoids with multiple cell lines. Three-dimensional (3D) CCA organoids include multiple cell lines, such as CCA cell lines (cholangiocytes; CHOL), liver sinusoidal endothelial cells (LSECs), and hepatic stellate cells (HSCs) (representative). Other liver cells, such as hepatocytes and Kupffer cells, can also be added, if needed. These organoids can be made scaffold-free without any biomaterial (eg, Matrigel) in a low-binding plate using culture mediums and can be kept for 14 to 28 days. Evidence suggests that CCA cells express cytokeratin (CK)-7, a cholangiocyte marker, and organoids can be stained with other cellular markers (eg, vascular endothelial cadherin; VE-Cad) that are present in organoids. CCA organoids can be exposed to immune cells, as seen at the bottom right. Bright-field microscopy shows a 3D CCA organoid exposed to mast cells. Scale bar = 500 μm. Original magnification, ×10.

The establishment of well-characterized CCA cell lines with a close resemblance to primary tumors, especially in a 3D microenvironment, will provide an infinite capacity for replicability, limiting the use of small animal in vivo studies and making them prime materials for CCA research.

Author Contributions

A.I. drafted the manuscript. A.Y., D.S., and E.A. wrote and reviewed the manuscript; B.E. developed the concept and wrote and critically reviewed the manuscript. All of the authors approved the final version.

Footnotes

Partially supported by an ASTS Faculty Development grant (B.E.); Indiana University Health Values Fund for Research award VFR-457-Ekser (B.E.); an Indiana University Health Foundation Jerome A. Josephs Fund for Transplant Innovation grant (B.E.); a Hickam Endowed Chair, Gastroenterology, Medicine, Indiana University grant (G.A.); an Indiana University Health–Indiana University School of Medicine Strategic Research Initiative grant (G.A.); Senior Career Scientist award IK6 BX004601 (G.A.); VA merit award 5I01BX000574 (G.A.); US Department of Veteran's AffairsCareer Scientist AwardIK6BX005226 (H.F.); VA merit award 1I01BX003031 (H.F.); Biomedical Laboratory Research and Development Service NIH grants DK108959 (H.F.), DK119421 (H.F.), DK054811 (G.A. and S.G.), DK115184 (G.A. and S.G.), DK076898 (G.A. and S.G.), DK107310 (G.A. and S.G.), DK110035 (G.A. and S.G.), DK062975 (G.A. and S.G.), and AA028711 (G.A. and S.G.); a Partners Seeking a Cure grant (G.A.); CPRIT grant RP210213 (S.C.); and the Richard L. Roudebush VA Medical Center (Indianapolis, IN).

Disclosures: None declared.

The views expressed in this article are those of the authors and do not necessarily represent the views of the US Department of Veterans Affairs.

References