<em>In Situ</em> Detection of Metabolically Active Cells in Hepatocellular Carcinoma Tissue Using MTT-Based Cryosection Imaging

Emerging evidence, including prior studies, highlights a subpopulation of cells within hepatocellular carcinoma (HCC) tissues that exhibit superior metabolic activity and stress resistance. These cells are key drivers of tumor progression and therapy resistance. However, existing methods for in situ viable cell detection often compromise tissue integrity, alter cellular viability, or are technically demanding. This study presents a novel, simple, non-destructive approach for in situ detection of metabolically active cells in HCC tissues through mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to insoluble formazan crystals. By optimizing tissue fragment size (1 × 1 × 0.2 cm³), culture conditions (20% FBS, 1 mg/mL MTT, and 3 h incubation with intermittent swirling), and cryosectioning parameters (20 µm thickness, 4% PFA fixation, and DAPI counterstaining), spatial mapping of viable HCC cells was achieved within 5 h. This cost-effective protocol requires no specialized equipment and maintains tissue architecture, enabling spatial identification of high-viability and stress-resistant cell clusters in surface regions. Limitations include progressive viability loss beyond 6 h and crystal displacement during sectioning, which can be mitigated through FBS supplementation and controlled freezing. This method provides a practical platform for single-cell isolation of therapy-resistant subpopulations, advancing HCC microenvironment research.