Table 2.
Isolation methods for various stromal cells in individual studies.
| Cell/ Cancer type | Dissociation | Incubation | Purification/Identification | Ref | ||
|---|---|---|---|---|---|---|
| Mechanical | Chemical/Enzymatic | Media | Substrate | |||
| Cancer-associated fibroblast (CAF) | ||||||
| Head and neck cancer | Not used | 10 mg/ml Collagenase P (2 hr, 37 °C) |
Fibroblast growth medium, 20% FCS, 1 ng/ml BFGF, 5 µg/ml Insulin, 1% P/S, 1% Gentamycin |
Tissue culture flask |
- VIM + αSMA + CD45- CD68- MelanA- HMB45- KRT- CD34- - Identified by flow cytometry and immunocytochemistry |
93 |
| Melanoma |
- Mincing - Scalpel - 1–4 mm2 |
0.25% Trypsin + 0.02% EDTA (20 min, 37 °C) |
High glucose DMEM, 10% FBS, 100U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml gentamycin |
6 well plate |
- Fibroblast: VIM - Myofibroblast: αSMA - Immunofluorescence - Minimize keratinocyte contamination by earlier detachment of fibroblasts after trypsin treatment |
78 |
| Breast cancer | - Dissection | Not used |
DMEM, 20% FBS, 1% Nonessential amino acid |
Cell culture plate |
- Spidle shaped morphology - Fibroblasts outnumbered tumor cells, and epithelial cells disappeared after passage 3 |
151 |
| Gastric cancer |
- Mincing - Scissors, scalpel - 2–4 mm |
Enzyme H, R, A of tumor dissociation kit (from Miltenyi Biotec) | RPMI1640, 10% FBS | Cell culture dish |
- CD90 - Morphology and FACS |
152 |
| Colorectal cancer |
- Mincing - 1 mm |
1 mg/ml Collagenase type I (1 h, 37 °C) | EGM2-MV | Tissue culture flask | - Proteomic and secretome analysis | 153 |
| Colorectal cancer |
- Mincing - 1 mm3 |
1.5 mg/ml Collagenase IV + 20 µg/ml Hyaluronidase (1 h, 37 °C) | DMEM, 10% FBS | Cell culture dish |
- 40 µm cell strainer - Red blood cell lysis buffer treatment - Wash away nonadherent cells after 3 h incubation - αSMA, VIM, and FAP - Immunofluorescence |
154 |
| Pancreatic cancer |
- Mincing - Scissors, scalpel |
Not used | DMEM, 10% FBS, 1X Glutamax, 1X P/S | 12 well plate |
- αSMA, VIM - myCAF: αSMA - iCAF: IL6 - apCAF: MHCII - Immunofluorescence |
155 |
| Liver cancer | - Cross sectioning | Enzyme H, R, A of tumor dissociation kit (from Miltenyi Biotec) | DMEM/F12 (1:1), 10% FBS | Cell culture dish |
- FAP + αSMA + EpCAM- ECad- PECAM1- - Spindle-shaped morphology and FACS |
156 |
| Ovarian cancer |
- Mincing - Scissors, scalpel - 3–5 mm2 |
- 0.25% Trypsin + 0.1% EDTA (30 min, 37 °C) - 0.5 mg/ml Hyaluronidase + 3 mg/ml Collagenase type 3 (6 h, 37 °C) |
RPMI1640, 20% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin | Cell culture dish |
- αSMA, VIM, FAP, PDGFRα, Desmin, DDR2, S100A4 - Mitotic figure - Immunofluorescence |
157 |
| Tumor-associated macrophage (TAM) | ||||||
| Breast cancer |
- Chopping - razor blade |
14 U/ml LiberaseTL + 28U/ml LiberaseDL + 15 mg/ml DNase I (1–18 h, 37 °C) | No cultivation | No cultivation |
- 100 µm cell strainer - CD45 + CD11b + CD14 + CD163 + CD3- CD19- CD56- - FACS |
158 |
| Breast cancer |
- Mincing - Scalpel - 2–4 mm |
0.1% Collagenase type I + 0.2% Dispase type I + 1% DNase I (30 min, 37 °C) | DMEM, 10% FBS, 100 units/ml penicillin, 10 µg/ml streptomycin | 96 well plate |
- 70 µm cell strainer - Red blood cell lysis buffer treatment - CD11b + F4/80+ |
159 |
| Breast cancer | - Mincing | LiberaseDH + DNase I (2 h, 37 °C) | DMEM, 5% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin | 24 well plate | - CD11b + F4/80+ | 160 |
| Breast cancer | Not used | Collagenase IV + Hyaluronidase + Dispase II + DNase IV (15 min, 37 °C) | DMEM | Cell culture plate | - Isolate CD11b + Ly6G- cells | 161 |
| Breast cancer |
- Mincing - razor blade |
0.29 U/ml LiberaseDL + 0.56 U/ml LiberaseTL + 150 µg/ml DNase I (45 min, 37 °C) | No cultivation | No cultivation |
- 100 µm cell strainer - Red blood cell lysis buffer treatment - DAPI- CD45 + CD11b + CD14 + CD163 + CX3CR1 + HLA-DR + CD3- CD19- CD56- - FACS |
79 |
| Mammary carcinoma |
- Cutting, mincing - razor blade |
1X Collagenase + 1X Hyaluronidase + 10U/ul DNase I (45 min, 37 °C) |
RPMI, 20% FBS, 2 mM L-glutamine, 2 mM Sodium pyruvate, 1X Penicillin‒streptomycin, 55 µM 2-mercaptoethanol, 10 ng/ml mouse M-CSF |
10 µg/ml Fibronectin-coated glass bottom plate |
- Isolate CD11b+ cells by MACS - Determine purity of F4/80 + CD45+ cells by FACS |
91 |
| Colorectal cancer | - Slicing | Accumax (2 h, 37°C) | DMEM, 10% FBS | 6 well plate | - CD68 | 162 |
| Ovarian cancer | Not mentioned | Not mentioned | RPMI 1640, 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin | Cell culture plate |
- Isolate CD14+ cells by MACS - Identified with CD206high HLA-DRlow by FACS |
163 |
| Mouse solid tumor |
- Mincing - Scissors, scalpel - 1–1.5 mm |
10 U/ml Collagenase I + 400 U/ml Collagenase IV + 30 U/ml DNase I (25 min, 37 °C) |
RPMI 1640, 10% FCS, 300 µg/ml L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.02 mM beta-mercaptoethanol, 1 mM sodium pyruvate, 1 mM nonessential amino acids |
No cultivation |
- 70 µm filter - Erythrocyte lysis buffer treatment - CD11b + Ly6G- SiglecF- Ly6Clow - MACS, FACS |
95 |
| Tumor endothelial cell (TEC) | ||||||
| Mammary cancer |
- Mincing - Scissors - 5 mm |
2 mg/ml Collagenase + 1 mg/ml Dispase + 1 mg/ml DNase (75 min, 37 °C) | Low glucose DMEM, 10% FBS, 10% Nu-Serum IV, 1% antibiotic-antimycotic, hFGF, VEGF, hEGF, R3-IGF-1, heparin | 0.5% Gelatin-coated cell culture dish |
- Scrape off nonspecific cells surrounding the identified EC colonies - Isolate CD31+ cells by MACS - Identified with CD31+ by immunofluorescence and FACS |
94 |
| Melanoma |
- Cutting - Scalpel - 1 mm3 |
10 mg/ml Collagenase type II + 25 µg/ml DNase I (30 min, 37 °C) | Endothelial cell growth medium MV2 | Collagenase type I- coated cell culture dish |
- Density gradient centrifugation - Isolate CD31+ cells by MACS - Determine the purity and sort cells with CD31 + CD146+ by FACS |
164 |
| Melanoma |
- Cutting - Scissors - 5 mm3 |
Collagenase type II + Collagenase type IV + DNase I (30 min, 37 °C) | No cultivation | No cultivation |
- 70 µm cell strainer - Isolate CD45- EpCAM- CD31+ cells by MACS |
165 |
| Melanoma | - Mincing | Collagenase II | EGM-2MV, 15% FBS | 1.5% gelatin-coated cell culture dish |
- Remove blood cells by single sucrose step-gradient centrifugation - Isolate CD31+ cells by MACS - Treatment of 500 ng/ml Diphtheria toxin to eliminate remaining tumor cells - Further purification with FITC-BS1-B4-lectin by FACS |
166 |
| Lung carcinoma |
- Mincing - Scalpel - 0.1 cm3 |
0.5 mg/ml Collagenase type I, 60 min, RT | Endothelial cell medium | Cell culture dish and Matrigel bed | - Isolate CD105+ cells by MACS | 167 |
| Renal carcinoma |
- Mincing - Scissors |
Collagenase type II (1 h, 37 °C) |
MCDB131 medium, 10 ng/ml EGF, 1 µg/ml Hydrocortisone, bovine brain extract, 20% FCS |
1% Gelatin or Endothelial cell attachment factor |
- CD105+ cells by MACS - Identified with CD105, CD31, and VWF by immunofluorescence and FACS |
168 |
| Cancer-associated adipocyte (CAA) | ||||||
| Breast cancer | Not used | 250 U/ml Collagenase type I (30 min, 37 °C) |
2D: DMEM, 10% FCS, 1% P/S 3D: DMEM, 25 mM glucose, 10% FBS, 1% P/S |
2D: 6 well plate 3D: 6 or 4.2 mg/ml fibrin matrix |
- 200 µm cell strainer - Wash with Krebs-Ringer Bicarbonate buffer for purification - Identified with Bodipy 493/503 by immunofluorescence |
169 |
| Breast cancer | - Mincing | 1 mg/ml Collagenase I (1 h, 37 °C) | DMEM/F12, 10% FBS | Cell culture plate |
- 100 µm cell sieve - Centrifugation |
170 |
| Omentum cancer |
- Mincing - Scissors, scalpel - mm size |
0.2% Collagenase I (1 h, 37 °C) |
DMEM/F12 (1:1), 1% Penicillin, 1% Streptomycin, 0.1% BSA |
Cell culture flask |
- Filter using nylon mesh - Identified with Bodipy 493/503 by immunofluorescence |
171 |
| Tumor pericyte | ||||||
| Hemangioma |
- Mincing, homogenize - Scalpel, pestle - 2 mm3 |
50 µg/ml LiberaseTM + 5 U/ml Dispase (40–50 min, 37 °C) | EGM2-medium, 10% hiFBS, 1% GPS, EGM-2 Single Quot supplements except hydrocortisone | 1 µg/cm2 FN-coated cell culture dish |
- 100 µm cell strainer - Isolate GLUT1- CD31- PDGFRβ+ cells by MACS |
172 |
| Colon carcinoma | - Mincing | Collagenase IA + Collagenase II + Collagenase IV + DNAse I (15–20 min, 37 °C) | No cultivation | No cultivation |
- 70 µm cell strainer - Isolate CD45- CD31- NG2 + CD140b+ cells by FACS |
173 |
| CRC/Lung cancer/Glioblastoma |
- Cutting - Scissors |
1.5 mg/ml Collagenase I + 1.5 mg/ml Collagenase II (40-60 min, 37 °C) | No cultivation | No cultivation | - Isolate NG2+ cells by MACS | 174 |
bFGF basic fibroblast growth factor, CD3 CD3 molecule, CX3CR1 C-X3-C motif chemokine receptor 1, DAPI 4′ 6-diamidino-2-phenylindole, DDR2 discoidin domain receptor tyrosine kinase 2, DMEM Dulbecco’s modified eagle medium, ECad E cadherin (CDH1), EDTA ethylenediaminetetraacetic acid, EGF epidermal growth factor, EGM2-MV endothelial cell growth medium MV2, EpCAM epithelial cell adhesion molecule, FACS fluorescence-activated cell sorting, FAP fibroblast activation protein alpha, FBS fetal bovine serum, FCS fetal calf serum, GPS guinea pig serum, hEGF human epidermal growth factor, hFGF human fibroblast growth factor, hiFBS heat inactivated fetal bovine serum, HMB45 premelanosome protein (PMEL), IL6 interleukin 6, KRT keratin, Ly6G lymphocyte antigen 6 family member G, M-CSF macrophage colony stimulating factor, MHCII major histocompatibility complex, class II (HLA-DR), NG2 neural/glial antigen 2, P/S penicilin/streptomycin, PDGFRα platelet derived growth factor receptor alpha, PECAM1 platelet and endothelial cell adhesion molecule 1 (CD31), R3-IGF-1 long Arg3 insulin like growth factor 1, RPMI1640 Roswell park memorial institute, S100A4 S100 calcium binding protein A4, VEGF vascular endothelial growth factor, VIM vimentin, VWF von willebrand factor, αSMA alpha smooth muscle actin.