Table 2 Various formulas of culture for different sample-derived CSCs and CTCs

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)

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

[83, 84]

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)

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)

[56]

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

14 d

[87]

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)

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)

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)

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;

14 d

[17, 33, 88,89,90,91,92]