Best Practices and Progress in Precision-Cut Liver Slice Cultures
Abstract
:1. Introduction
2. Precision-Cut Liver Slice Parameters
2.1. Tissue Slicer
2.2. Liver Slice Thickness
2.3. Liver Slice Diameter
2.4. Liver Slice Culture Conditions
2.5. Species
2.6. Disease Model
3. Precision-Cut Liver Slice Read-Outs
3.1. Culture Assessment
3.1.1. Viability and Cytotoxicity
Read-Out | Advantages | Disadvantages/Difficulties | Analysis Time | Cost per Data Point |
---|---|---|---|---|
Viability and Cytotoxicity | ||||
LDH leakage | Slice specific follow-up over time High throughput | Reader required | ±1 h | € |
ATP assay | High throughput | End point analysis only Total tissue lysis required Reader required | ±1 h | € |
Morphology | Evaluation of liver structure | Interpretation not always straightforward Embedding technically challenging | ±3 h | € |
Slice analysis | ||||
qRT-PCR m(i)RNA analysis | Evaluation of multiple m(i)RNA’s | Selective m(i)RNA measurements | ±3 h | €€ |
Gene expression profiling | Generation of unbiased and complete mRNA profile | Need for bioinformatic skills Need for (nucleomics) infrastructure | Several days | €€€€ |
Western Blot | Medium sensitivity High specificity | Low throughput High tissue input required | 1 day | €€€ |
Hydroxyproline assay | Quantitative | High tissue input required Accurate measurement of input | 1 day | €€ |
Immunohistochemical stainings | Can provide more detailed insight into zonation and cell type specific protein expression | Embedding technically challenging Relative low throughput Limited amount of stainings per slice | >1 day | €€ |
Triglyceride quantification | Fatty/metabolic liver disease specific | Total homogenization of tissue required | ±2 h | €€ |
GSH content | Functional | Affected by fat storage [46] | ±1 h | €€ |
Medium analysis | ||||
ALT, AST measurements | Straightforward Direct indicator of liver damage | Concentration of medium might be required/sufficient level of damage required to exceed threshold Low throughput | Minutes | € |
ELISA | Quantitative Multiple proteins can be tested for 1 slice | Low throughput | Hours | €€€ |
Protein Profiling | Generation of unbiased and complete protein profile | Need for bioinformatic skills Need for (proteomics) infrastructure | Several days | €€€€ |
3.1.2. Morphology
3.2. Slice Analysis
3.2.1. RNA Analysis
3.2.2. Protein Analysis
3.2.3. Other Tissue Analyses
3.3. Medium Analysis
3.4. Other Applications
Liver Disease | Recommended Read-Outs |
---|---|
Toxicology | Hepatocyte viability by LDH leakage or ATP assay [86] Hepatocyte viability by functionality through Albumin ELISA [73] |
Fibrosis | LDH leakage or ATP assay (hepatocyte viability) [71] mRNA levels of fibrosis markers by qRT-PCR [24] Protein levels of fibrosis markers (WB, staining or ELISA) [24] Sirius Red staining for cross-linked collagen [24] |
Fatty liver disease | LDH leakage or ATP assay [58] Lipid Staining (e.g., ORO) [58] Triglyceride analysis/quantification [58] qRT-PCR for fibrosis mRNA markers [87] |
Alcoholic liver disease | LDH leakage or ATP assay [42] Medium analysis for metabolites [42,88] qRT-PCR for fibrosis mRNA markers [42,85] Electron microscopy [65] * |
Inflammation/Immunity | ELISA on medium for inflammatory markers [48,72] qRT-PCR for inflammatory mRNA markers [89] |
Cholestasis | LDH leakage or ATP assay [90,91] qRT-PCR for cholestatic mRNA markers [91] Bile acid determination [91] |
Cancer | LDH leakage or ATP assay [21] qRT-PCR for cancer-related mRNA markers [21] H&E staining (tumor morphology) [54] Staining for Ki67 (proliferation) [54] |
4. Future Perspectives
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Dewyse, L.; Reynaert, H.; van Grunsven, L.A. Best Practices and Progress in Precision-Cut Liver Slice Cultures. Int. J. Mol. Sci. 2021, 22, 7137. https://doi.org/10.3390/ijms22137137
Dewyse L, Reynaert H, van Grunsven LA. Best Practices and Progress in Precision-Cut Liver Slice Cultures. International Journal of Molecular Sciences. 2021; 22(13):7137. https://doi.org/10.3390/ijms22137137
Chicago/Turabian StyleDewyse, Liza, Hendrik Reynaert, and Leo A. van Grunsven. 2021. "Best Practices and Progress in Precision-Cut Liver Slice Cultures" International Journal of Molecular Sciences 22, no. 13: 7137. https://doi.org/10.3390/ijms22137137