Here, we present a simple protocol to detect the metabolically active cells in hepatocellular carcinoma tissue using MTT-based cryosection imaging. This method may also be used to detect active cells in situ in other tissues or organs.
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Method Article
* These authors contributed equally
Here, we present a simple protocol to detect the metabolically active cells in hepatocellular carcinoma tissue using MTT-based cryosection imaging. This method may also be used to detect active cells in situ in other tissues or organs.
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.
Hepatocellular carcinoma (HCC), the third leading cause of cancer-related deaths globally, is characterized by high heterogeneity and recurrence rates1. This spatial and temporal heterogeneity makes certain cell populations within tumor tissues more active than others in metabolism, proliferation, progression, stress tolerance, and drug resistance. The in situ detection of viable cells within HCC tissues holds profound clinical and biological significance, as these cells encompass proliferating subclones, therapy-resistant populations, and metastasis-initiating cells. Such viable cells could serve as critical drivers of tumor recurrenc....
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All experiments were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the First Affiliated Hospital of Quanzhou (No. FJMU IACUC2021257). HCC tissues were surgically removed and collected at Quanzhou First Hospital, affiliated to Fujian Medical University, with informed consent from the patients. This experiment requires standard protective measures, including wearing a laboratory coat, a disposable mask, and gloves.
1. Tissue preparation
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This study successfully detected metabolically active cells in HCC tissue without disrupting tissue architecture. Firstly, the brightfield images (Figure 1A) showed that the tissue surface exhibited dense cellular coverage and multilayered stacking of cells across the slide, confirming the preserved architectural integrity of the HCC tissue throughout culture and processing. Notably, clusters of cells within superficial regions displayed distinct purple-red formazan deposits, indicative of m.......
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Current methodologies for in situ viable cell detection still face multifaceted limitations. IHC and immunofluorescence (IF), which are widely used to identify proliferation markers or apoptosis-related proteins, suffer from disruption of membrane integrity induced by formaldehyde fixation7. Moreover, antibody penetration in thick tissue sections is suboptimal8. Fluorescent viability probes such as Calcein-AM, though effective in labeling esterase-active live cells.......
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The authors have no conflicts of interest to disclose.
This research was funded by the Science and Technology Bureau of Xiamen City (No. 3502Z20227197), Fujian Medical University's Startup Fund for Scientific Research (2021QH1240). We thank Xianying Zhang, Jingjing Zhou, and Shaocong Weng (Huaqiao University) for their technical assistance.
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| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| -80 °C freezer (upright) | Thermo Fisher Scientific | TSX60086V | |
| Clean bench (vertical laminar flow) | AIRTECH | SW-CJ-1FD | |
| CO2 incubator (165 L) | Thermo Fisher Scientific | Heracell VIOS 160i (e.g., 51030400 / 51033559) | |
| Coverslip (24×50 mm, #1.5) | CITOGLAS (Citotest) | 0341-3610 | |
| Culture petri dish (90 mm) | NEST | 752001 (example) | |
| DAPI-containing mounting medium | Beyotime | P0131 (5 mL/25 mL) | |
| DMEM (high glucose, w/ pyruvate) | Invitrogen (Gibco) | 11995-065 | |
| FBS (Qualified, US origin) | Invitrogen (Gibco) | A3160501 | |
| Fluorescent microscope | Nikon | ECLIPSE Ti2 series | |
| Freezing microtome (cryostat) | Leica | CM1950 | |
| Microscope slides (plain, 25×75 mm) | CITOGLAS (Citotest) | 0317-0001 | |
| MTT reagent (powder) | Beyotime | ST316 | |
| Nail polish (clear) | Beyotime | — | |
| NBF (Neutral Buffered Formalin) | Beyotime | — | |
| OCT compound | SAKURA (Sakura Finetek) | 4583 | |
| Optical microscope (upright) | Nikon | ECLIPSE Ci series (e.g., Ci-L) | |
| PBS (500 mL) | Beyotime | C0221A | |
| PFA, 4% in PBS (fixative) | Beyotime | P0099 | |
| Pipette tip (1 mL, low-binding) | KIRGEN | KG1313-L | |
| Pipette tip (200 µL) | Axygen (Corning) | T-200-Y (bulk) / T-200-Y-R-S (racked, sterile) | |
| Pipettor (single-channel) | Thermo Fisher Scientific | Finnpipette F2 (range-specific cat#) | |
| Scalpel | HYSTIC | model varies (e.g., supplier ref. 100022208326) | |
| Tweezer | HYSTIC | model varies |
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