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Premade AAV, adenovirus, lentivirus particles, safe, stable, in stock.
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Human Druggable Genome siRNA Library enables efficient drug target screening.
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Chromogenic LAL Endotoxin Assay Kit ensures precise, FDA-compliant endotoxin quantification for biosafety testing.
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Powerful Tn5 Transposase for DNA insertion and random library construction.
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Aptamers for key proteins like ACVR1A, Akt, EGFR, and VEGFR.
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Harness the power of protein degraders for precise protein degradation, expanding druggable targets and enhancing therapeutic effectiveness for cutting-edge drug discovery.
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3D in vitro cancer cell models represent a bridge between cell lines and tumors grown in vivo. They effectively mimic the 3D characteristics of solid tumors with heterogeneous access to nutrients and oxygen. This protocol focuses on tumorsphere culture, where cells are grown in serum-free media on ultra-low attachment plates, allowing the enrichment of cancer cells with stem/progenitor properties.
Table 1. Advantages and Disadvantages of Different 3D Cell Culture Techniques. (Fang Y, et al., 2017)
Ultra-low attachment plates (6-well and 24-well, Corning)
70 µm cell strainer (Corning, Falcon®)
DMEM/F12, Glutamax (Gibco)
B27 supplement (50x, serum-free, Gibco)
bFGF and EGF (Gemini Bio-Products)
ROCK inhibitor (Y-27632, STEMCELL Technologies)
Matrigel or Cultrex Matrix (Growth Factor Reduced)
Collagenase IV (Sigma-Aldrich)
Dispase (1 U/ml, STEMCELL Technologies)
Accutase (STEMCELL Technologies)
Antibiotic-Antimycotic (100x, Gibco)
Mission pLKO.1-puro-CMV-TurboGFP Positive control Transduction Particles (optional)
SureEntry transduction reagent (QIAGEN)
Trypan Blue (Bio-Rad)
DMSO (Sigma-Aldrich)
At Creative Biogene, we offer top-tier tumor cell lines designed to advance your cancer research. Our cell lines are meticulously developed to replicate the 3D characteristics of solid tumors, providing a robust model for studying tumor biology and testing potential treatments.
Cell Transduction with shRNA
1. Prepare cells at 60-70% confluence.
2. Detach cells with Trypsin, and wash with PBS containing SureEntry reagent (8 µg/ml).
3. For each transduction, use 50,000 cells and 10 µl of high-titer virus (109 TU/ml).
4. Mix cells and virus in an Eppendorf tube, and transfer to a 50 ml Falcon tube.
5. Incubate for 30 min at 37°C, 5% CO2.
6. Add 2-3 ml culture media and plate in a 6-well plate or 10 cm dish.
7. Change the medium the next day and allow recovery for 2-4 days.
8. Add selection antibiotic for 12-14 days.
9. Verify knockdown efficiency by qPCR or Western blot.
3D Spheroid Culture (Matrigel-embedded)
1. Detach cells and filter through a 70 µm strainer.
2. Count cells and resuspend in a cold CSC medium.
3. Mix with ice-cold Matrigel (1:3 ratio Matrigel to media).
4. Plate on ultra-low attachment plates (5,000 cells/well for 24-well, 10,000 cells/well for 6-well).
5. Incubate overnight to allow Matrigel solidification.
6. Gently add CSC medium the next day; replenish every 3-5 days.
Figure 1. Tumorspheres cultured in CSC media alone (A) or embedded in Matrigel (B). Tumorspheres in CSC media show a tendency to clump. Scale bars: 50 µm.
Primary 3D Spheroid Culture from Tumor Tissue
1. Obtain fresh tumor tissue in sterile PBS or Hibernation medium.
2. Wash 2-3x with PBS + Antibiotic-Antimycotic.
3. Mince tissue into 1-3 mm pieces and digest with 0.5-1% Collagenase IV.
4. Monitor digestion every 15 min, checking cell viability.
5. Wash out collagenase and filter through a 70 µm strainer.
6. Count cells and plate up to 0.5 x 106 cells per well (6-well plate) in CSC medium or CSC medium + Matrigel.
7. Monitor growth daily and passage as needed.
Passaging Spheroids
1. Collect Matrigel-embedded spheroids.
2. Add 10 ml of Dispase (1 U/ml) and incubate for 30-60 min at 37°C.
3. Stop the reaction with 0.005 M EDTA and wash with PBS.
4. For single-cell suspension, treat with 5 ml Accutase.
5. Monitor digestion and resuspend to single cells when ready.
Cryopreservation
1. Prepare single cells (0.5-2 x 106) or tumorspheres from one well of a 6-well plate.
2. Resuspend in 1 ml CSC medium + 10% DMSO.
3. Freeze in Mr. Frosty container at -80°C, then transfer to the liquid nitrogen vapor phase.
Data Analysis
1. Image spheroids using confocal microscopy.
2. Take 5-20 fields per well, ensuring consistent imaging between samples.
3. Measure spheroid diameters using ImageJ software:
- Open image in ImageJ
- Use the "Oval" selection tool to measure the diameter
- Calibrate measurements using a scale bar
4. Analyze data using GraphPad Prism or R software:
- Plot diameter distributions
- Perform statistical analysis (e.g., Wilcoxon test for non-normal distributions)
- Compare proportions of spheroids vs. single cells/clumps
1. Sterilize fresh tumor tissue with 70% ethanol for 3-5 seconds before processing.
2. Optimize cell seeding density for each cancer cell type.
3. Monitor spheroid formation daily and gently resuspend if clumping occurs.
4. For primary cultures, consider adding 1% FBS if initial growth is poor.
5. When passaging, be careful not to disturb the Matrigel layer containing spheroids.
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