In Situ Detection of Metabolically Active Cells in Hepatocellular Carcinoma Tissue Using MTT-Based Cryosection...

Area of Science:

• Oncology
• Cell Biology
• Biochemistry

Background:

• Hepatocellular carcinoma (HCC) contains a subpopulation of cells with high metabolic activity and stress resistance.
• These cells drive tumor progression and therapy resistance.
• Current in situ viable cell detection methods often compromise tissue integrity or are technically complex.

Purpose of the Study:

• To develop a simple, non-destructive method for in situ detection of metabolically active cells in HCC tissues.
• To enable spatial mapping of viable HCC cells and identify therapy-resistant subpopulations.

Main Methods:

• Utilized mitochondrial-dependent reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan crystals for viable cell detection.
• Optimized tissue fragment size, culture conditions (20% FBS, 1 mg/mL MTT, 3h incubation), and cryosectioning parameters (20 µm thickness, 4% PFA fixation, DAPI counterstaining).

Main Results:

• Achieved spatial mapping of viable HCC cells within 5 hours using the optimized protocol.
• The method is cost-effective, requires no specialized equipment, and preserves tissue architecture.
• Identified high-viability and stress-resistant cell clusters in surface regions of HCC tissues.

Conclusions:

• The developed protocol offers a practical platform for in situ detection and spatial mapping of viable cells in HCC.
• Enables single-cell isolation of therapy-resistant subpopulations for further research into the HCC microenvironment.
• Addresses limitations of existing methods by maintaining tissue integrity and cellular viability.