Background
The viable CTCs with CSCs feature for functional analysis
Functional analysis of CTCs revealed modern individual treatment
The expanded methods of CTCs for clinical individual application
Method | CDX | Short term | Long term |
---|---|---|---|
CTC number | High | Low | High |
Patient origin | Advanced stage only | Early and advanced stage | Advanced stage only |
Condition | Experimental animal | 10% FCS medium | Defined serum-free medium |
Sample origin | Organ-vasculature circulation | Peripheral venous or arterial circulation | Peripheral venous or arterial circulation |
Character | Tumorigenic capacity evaluation; complex procedure and individual difference | Differentiation and limited proliferation ability with significant phenotypic alterations | Phenotype stable; maintaining the tumorigenicity in non-adherent status |
Research purpose | Simulate microenvironment in vivo | Expand enough CTCs for downstream analyses | Enrich and expand CTCs to establish patient-derived cell lines for long-term research |
Cost | High | Cheap | Moderate |
Culture cycle | Several months | 1-2 weeks | Several months −1 year |
Successful rate | Low | Moderate | Low |
Optimize the current approaches of CTCs culture
Purpose | Cell origin | Culture material | Cell seeded concentration | Initial treatment | Medium | Added ingredients | Environment | Culture cycle | ref |
---|---|---|---|---|---|---|---|---|---|
CSC/SC Sphere formation | Bladder cancer cells | Ultra-low attachment surface (Corning) | 6 × 103 cells/well | 6-well plates | Serum-free DMEM/F12 (Gibco) | 20 ng/mL EGF(Invitrogen), 20 ng/mL bFGF (Invitrogen), 1% N2 (Invitrogen), 2% B27 (Invitrogen) and 1% penicillin-streptomycin (Hyclone) | 2 w | [79] | |
Pancreatic Cancer KPCL Cell Line | Ultra-low attachment plates (Corning) | Tumor tissue minced | Promote organoid formation in serum-free for 3 days | Serum-free DMEM/F12 | 0.5% methylcellulose, 1% N2 (Invitrogen), 2% B27 (Invitrogen), 20 ng/ml recombinant human EGF (Miltenyi Biotec) and 20 ng/ml recombinant human FGF-2 (Miltenyi Biotec), 5 μg/ml heparin (Sigma) and 1% penicillin/streptomycin (Invitrogen) | 3 d | [80] | ||
Kidney cancer cell lines ACHN /CAKI-1 RCC | Ultra-low attachment plates (Corning) | 500 cells/well | 96-well plate; 100 μl SFDM/well; add 25 μl SFDM /well /day | Serum-free defined media (SFDM) low-glucose (1 g/l) DMEM | L-Glutamine, sodium pyruvate, Penicillin/Streptomycin (Wisent Inc), 20 ng/ml basic FGF, 20 ng/ml EGF, and B27 (Invitrogen, Grand Island, USA) | 3 w | [81] | ||
Brain metastases tumor | Ultra-low attachment surface (Corning) | 200 to 2 cells /well (limiting dilution) | 100 μL of cNSC media in a 96-well plate | Complete NSC (cNSC) media | Complete NSC media is comprised of NSC basal media (1% N2 [Gibco], 0.2% 60 μg/mL N-acetylcystine, 2% neural survival factor-1 [Lonza], 1% HEPES, and 6 mg/mL glucose in 1:1 DMEM/F12 [Gibco]), supplemented with 1× antibiotic–antimycotic (Wisent), 20 ng/mL human epidermal growth factor (Sigma),20 ng/mL basic fibroblast growth factor (Invitrogen), and 10 ng/mL leukemia inhibitory factor (Chemicon) | 37 °C, 5% CO2 | 7 d | [82] | |
Mammary gland stem cells | Low-attachment culture plate (Corning) | Serial dilution; 5-2000/well | 96-well plate; | MM+ medium | DMEM/F12 supplemented with 2% calf serum, 10 mmol/L HEPES, 20 ng/mL epidermal growth factor (EGF), 10 μg/mL insulin, 5% bovine serum albumin, 1:50 B27 (Invitrogen), 20 ng/mL, basic fibroblast growth factor (bFGF), and 10 μg/mL heparin and 100 μg/mL penicillin/streptomycin | 7 d | [67] | ||
Breast organoids | 50-mm low attachment plat (Corning) | 2.5 × 105 cells/well | Dissociated into single cells after 6–8 h into 6-well plates | Serum free DMEM/F12 media | 10 ng/ml hEGF, 1 mg/ml hydrocortisone, 10 mg/ml insulin, 20 ng/ml bFGF, 4 ng/ml heparin (Sigma Aldrich), B27 (Invitrogen) supplemented with antibiotics | 7 d | |||
HCC1806/MCF10A | Ultra-low attachment plates(Corning) | 5 × 103 cells/well | Mammary epithelial growth medium (MEBM) | Serum-free mammary epithelial growth medium (MEBM) (Lonza), supplemented with B27 (Invitrogen), 20 ng/mL EGF and 20 ng/mL bFGF (BD Biosciences), and 4 μg/mL heparin (Sigma). | 10–14 d | [85] | |||
Brain tumor cell lines | Cells grown as monolayers were transfered into serum-free medium | DMEM high glucose (Sigma) | Serum free stem cell medium: DMEM/F12 (70/30%), 2% B27 (Invitrogen), 5 ng/mL heparin (Sigma), supplemented with 20 ng/mL human recombinant epidermal growth factor (hrEGF; Invitrogen), and 20 ng/mL human basic recombinant fibroblast growth factor (bFGF; BD Bioscience) | 37 °C, 5% CO2 | 4–5 w | [86] | |||
Gastric cancer cell (patient- derived) | A single cell in 96-well plate | Samples were subjected to mechanical /enzymatic dissociation | Neurobasal-A medium (Gibco, Camarillo, CA) | Neurobasal-A medium (Gibco, Camarillo, CA) supplemented with 2 mM L-glutamine, 120 lg/ml of penicillin, 100 lg/ml of streptomycin, B27, 50 ng/ml of EGF, and 50 ng/ml of FGF-2. For differentiation, 5% FCS was added to the media instead of growth factors. | 10 days | [68] | |||
PC3 human prostate cancer cells | 100 cm2 culture dishes | 1000 cells/ml | DMEM/F12 | Serum-free DMEM/F12 medium containing 20 ng/ml epidermal growth factor (EGF; R and D Systems, Minneapolis, MN), 5μg/ml insulin, 0.4% bovine serum albumin (Sigma, St. Louis, MO), and 2% B27 (Invitrogen, Carlsbad, CA) | 37 °C; humidified atmosphere; 5% CO2 | [56] | |||
CSC 3D culture | GBM6 cell line | 3D CHA scaffold culture | 50,000 cells /scaffold; 12-well plates | DMEM | DMEM supplemented with 2.5% FBS and 1% penicillin/streptomycin | 37 °C humidified atmosphere 5% CO2 | 14 d | [87] | |
CTC culture | Patients with metastatic CRC (stage IV) | Ultralow attachment plates (Corning) | N/A | In 24-well plates | M12 medium (1 mL/well) | M12 medium contains advanced DMEM/F12 (Gibco), 2 mmol/L of L-glutamine, 100 Unit/mL of penicillin and streptomycin, N2 supplement (Gibco), 20 ng/mL of epidermal growth factor (R&D) and 10 ng/mL of fibroblast growth factor-basic (R&D) | 3 w (5 × 106 cells) | [25] | |
Patients with breast cancer | N/A | 24- or 6-well plates for further growth, and subsequently into T75 tissue culture flasks | DMEM/F12 | Stem cell culture medium (DMEM/F12 containing 5 mg/ml insulin, 0.5 mg/ml hydrocortisone, 2% B27, 20 ng/ml EGF, and 20 ng/ml FGF-2) for the first seven days, then switched to EpiCult-C medium from day 8 (STEMCELL Technologies Inc.) with 10% FBS and 1% penicillin/streptomycin and continued to grow in this medium until day 21. The medium used from day 22 on was DMEM/F12 plus 10% FBS and 1% penicillin/streptomycin solution (Regular M) | 37 °C, 5% CO2, | 0-7;8-21;>22d | [77] | ||
Patients with colon tumor | N/A | DMEM/F12 | Sphere medium used was DMEM/F12- Heparin 0.5 U/ml, EGF 50 ng/ml, FGF 25 ng/ml, BSA 1%, penicillin–streptomycin solution 1%. | 14 d (short term) | [48] | ||||
Patients with colon tumor | Non adherent plates | N/A | Culture in 24 well and into T25 flasks for culture expansion | DMEM/F12; RPMI1640 | DMEM/F12 containing insulin (20 μg/mL), 1% N2 complement, epithelial growth factor (EGF: 20 ng/mL), L-Glutamine (2 mM), fibroblast growth factor-2 (FGF2: 10 ng/mL) and 2% foetal calf serum for the first days (Medium 1). After a few weeks, the CTC culture was switched to another appropriate culture medium to improve the CTC cell growth (Medium 2: RPMI1640, Growth factors: EGF and FGF-2, Insuline-Transferine-Selenium supplement, L-Glutamine) under normoxic conditions (5% CO2) | Hypoxic conditions; 2% O2; 37 °C | A few months obtained billions of tumor cells | [31] | |
Patients with lung cancer (early stage) | 3D material: Collagen; matrigel; fibroblasts | N/A | 3D co: a mix of collagen and matrigel and fibroblasts 3D mono: cultured only with gel; 2D co: cultured only with fibroblasts 2D mono: without any gel nor fibroblasts | RPMI1640 | RPMI1640 (10% FBS and 1% Penicillin/Streptomycin) maintained under different culture conditions and cultured up to 7 days on the chip: 3Dco; 3Dmono; 2Dco; 2Dmono | 14 d | [54] | ||
Patients with head and neck tumor | Non adherent spheroid microplates (Thermo Scientific, USA) | N/A | Isolated CTCs were cultured in 96F well | DMEM/F12 | Culture medium containing Advanced DMEM/F12 with the following additives: 50 ng/mL EGF (Sigma), 5% v/v R-spondin 1, 10% v/v Noggin, 10 ng/mL FGF10 (Peprotech), 1 ng/ml FGF2 (Peprotech), 10 nM Nicotinamide (Acros), 0.5 μM A83–01 (Tocris), 10 μM SB202190 (Sigma Aldrich), 10 μM Y-27632 (Selleck Chemical), 1X B27 Additive (Invitrogen), 1.25 mM N-Acetyl-L-cysteine (Sigma-Aldrich), 2 nM Glutamax(Invitrogen), 10 mM HEPES (Sigma Aldrich), 1:100 v/v Primocin (Invivogen) | 2% O2; 5% CO2; 37 °C | 14 d | [32] | |
Patients with pancreatic/urothelial/urinary bladder/ prostate Cancer | N/A | 6-well cultivation plate | RPMI1640 | Isolated CTCs by size-based separation methodMetaCell®, and grown in FBS-enriched RPMI1640 (10%) for a minimum of 3-6 days; | 37 °C, 5% CO2 | 14 d |