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Annorade mRNA Sequencing Qualified the Transcriptome Analysis of Fresh and Warmed Tissue of Hepatocellular Carcinoma

ABSTRACT: Purpose: The aim of this study was to create a patient-derived slice model by combining cryopreservation technique with precision-cut slice culture method and explore its effectivity of predicting anti-cancer drug sensitivity in vitro. Methods: We prepared 0.3 mm thick tissue slices by a microtome and maintain its cell viability by cryopreservation technique. Slices were cultured individually in the presence or absence of regorafenib (REG) for 72 hours. Alterations in morphology and gene expression were assessed by histological and genetic analysis. Overall viability was also analyzed in tissue slices by CCK-8 quantification assay and fluorescent staining. Tissue morphology and cell viability could be evaluated to quantify drug effects. Results: Histological and genetic analysis showed that no significant alterations in morphology and gene expression were induced by vitrification‑based cryopreservation. The viability of warmed HCC tissues was up to 90% of the fresh tissues. The viability and proliferation could be retained for at least four days in filter culture system. The positive drug responses in precision-cut slice culture in vitro were evaluated by tissue morphology and cell viability. Conclusion: In summary, the successful application of precision-cut HCC slice culture combining cryopreservation technique in a systematic drug screen demonstrates the feasibility and utility of slice culture method for drug response.

ORGANISM(S): Homo sapiens

PROVIDER: GSE194095 | GEO | 2022/01/21

REPOSITORIES: GEO

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Project description:Background: Evidence has recently accumulated suggesting that mature hepatocytes have the unique capacity to autonomously decide their replication fate. The molecular mechanism by which hepatocytes switch from an essentially quiescent state to rapidly proliferation after liver injury is not fully understood. To provide molecular insights into the self-renewal of mature hepatocytes, we have applied an ex vivo liver slice culture system. Results: After liver injury, activation of p38 and Erk1/2 began in mature hepatocytes within 5 min. Erk1/2 was activated at the edge of the cut as well as on the surface of liver slices. The number of hepatocytes that contain activated Erk1/2 increased within 1 h and then decreased. Concomitantly, immediate early genes (IEGs), such as Jun, early gene response 1 (Egr1) and Myc were induced and Ki67 positive hepatocytes appeared 24 h after liver injury in the slices. In agreement with in vivo studies, transcriptome analysis using 2 h slice culture reveals that 63% of upregulated genes were downstream of lipopolysaccharide (LPS) stimulation which induces interleukin 6 (IL 6) or tumor necrosis factor (TNF) alpha pathways. Furthermore, circadian clock regulator, such as members of the basic helix-loop-helix family, Bhlhe40 and Bhlhe41, were highly upregulated for 4 h after injury. Although upon injury Jun or Egr1 were induced in liver slices from proliferation-defective liver, no circadian clock regulator genes were upregulated. Moreover, using an in vitro cell culture system we show that Bhlhe40 is required for the G0-G1 transition. Conclusion: Our data suggest that the circadian clock regulator, Bhlhe40, is involved in the G0-G1 transition. An ex vivo system using normal and proliferation defective KO liver is a useful tool for identification of genes that trigger cell proliferation shortly after liver injury. This method may also be applied for measurement of the liver regeneration potential of individual livers at the priming phase. In one dual-color microarray hybridization, mRNA expression changes after 2h ex vivo incubation of liver slices were examined.

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Annorade mRNA Sequencing Qualified the Transcriptome Analysis of Fresh and Warmed Tissue of Hepatocellular Carcinoma

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