Scholarlink

<p>This work establishes a robust and reproducible 96-well plate-based 3D HepG2 tumour spheroid model for drug screening applications. A 7-d culture protocol was optimised to generate HepG2 spheroid model using 96-well ultra-low attachment plates with 5 seeding densities (500, 1000, 2500, 5000 and 10,000 cells/well). Spheroids were evaluated for morphology, viability and proliferation. The optimized model was treated with doxorubicin and sorafenib at their respective IC<sub>50</sub> values (2.4&#xa0;µM and 5.3&#xa0;µM, determined in HepG2 monolayer). Drug-specific responses were assessed via spheroid growth, viability and expression of apoptotic and drug resistance markers. Spheroids seeded at 500 cells/well exhibited optimal characteristics, including sustained proliferative capacity (up to 30-fold increase relative to day 1) and progressive spheroidal growth (1.85-fold-change in diameter); hence, it was selected for further experiments. Day 4 was identified as the optimal treatment point based on morphology, viability and diameter progression. The 72-h IC<sub>50</sub> values were determined to be 2.4&#xa0;µM for doxorubicin and 5.3&#xa0;µM for sorafenib; these concentrations were chosen for subsequent treatments on HepG2-derived spheroids. Doxorubicin induced a delayed but potent cytotoxic response, while sorafenib triggered a slower, sustained effect, distinct from its rapid action in monolayer cultures. Furthermore, spheroid diameter did not correlate with viability or proliferation decline, highlighting its limitation as a sole readout. Molecular profiling revealed drug-specific responses in which <i>ABCB1</i> was upregulated by doxorubicin, while <i>ABCC2</i> was selectively induced by sorafenib. Sorafenib progressively reduced <i>BAX/BCL2</i> ratio, suggesting potential resistance development. This study presents an optimized HepG2 spheroid model with validated culture and treatment parameters, capturing drug-specific cellular and molecular responses for therapeutic screening and mechanistic studies in hepatocellular carcinoma.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

3D HepG2 spheroid model exhibits drug-specific response profiles validating their application in therapeutic screening

  • 期刊论文

  • 发表日期：

    2026年7月21日

查看全文
  • Wee Ling Choe,
  • Ain Zubaidah Ayob,
  • Muhammad Danish Ahmad Tarmizi,
  • Nur’ain Salehen,
  • Thamil Selvee Ramasamy

摘要

This work establishes a robust and reproducible 96-well plate-based 3D HepG2 tumour spheroid model for drug screening applications. A 7-d culture protocol was optimised to generate HepG2 spheroid model using 96-well ultra-low attachment plates with 5 seeding densities (500, 1000, 2500, 5000 and 10,000 cells/well). Spheroids were evaluated for morphology, viability and proliferation. The optimized model was treated with doxorubicin and sorafenib at their respective IC50 values (2.4 µM and 5.3 µM, determined in HepG2 monolayer). Drug-specific responses were assessed via spheroid growth, viability and expression of apoptotic and drug resistance markers. Spheroids seeded at 500 cells/well exhibited optimal characteristics, including sustained proliferative capacity (up to 30-fold increase relative to day 1) and progressive spheroidal growth (1.85-fold-change in diameter); hence, it was selected for further experiments. Day 4 was identified as the optimal treatment point based on morphology, viability and diameter progression. The 72-h IC50 values were determined to be 2.4 µM for doxorubicin and 5.3 µM for sorafenib; these concentrations were chosen for subsequent treatments on HepG2-derived spheroids. Doxorubicin induced a delayed but potent cytotoxic response, while sorafenib triggered a slower, sustained effect, distinct from its rapid action in monolayer cultures. Furthermore, spheroid diameter did not correlate with viability or proliferation decline, highlighting its limitation as a sole readout. Molecular profiling revealed drug-specific responses in which ABCB1 was upregulated by doxorubicin, while ABCC2 was selectively induced by sorafenib. Sorafenib progressively reduced BAX/BCL2 ratio, suggesting potential resistance development. This study presents an optimized HepG2 spheroid model with validated culture and treatment parameters, capturing drug-specific cellular and molecular responses for therapeutic screening and mechanistic studies in hepatocellular carcinoma.