Related Experiment Video
Updated: Aug 26, 2025
Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs
Published on: December 16, 2022
Surprising efficacy twist of two established cytostatics revealed by a-la-carte 3D cell spheroid preparation protocol
Jiřina Kroupová1, Jaroslav Hanuš1, František Štěpánek1
1Department of Chemical Engineering, University of Chemistry and Technology, Prague, Technicka 3, 166 28 Prague 6, Czech Republic.
Abstract:
Three-dimensional cell culture systems are increasingly used for biological and anticancer drug screening as they mimic the structure and microenvironment of tumors more closely than conventional two-dimensional cell models. In this study, the growth kinetics of colon adenocarcinoma-derived spheroids (HT-29 cell line) cultivated in liquid marble micro-bioreactors and nonadherent PDMS-coated well plates was investigated in detail and enabled precise control of the spheroid size by the seed cell density and cultivation time. The therapeutic effect of 5-fluorouracil and irinotecan hydrochloride in 2D monolayer cell culture and 3D tumor spheroids revealed an unexpected twist in their efficacy due to different ability to penetrate through 3D microtissue. For 5-fluorouracil, the inhibitory concentration IC50 after 48 h exposure increased from 11.3 µM for a 2D cell culture to 707.7 µM for a 3D spheroid. In the case of irinotecan, IC50 increased from 24.9 µM to 77.8 µM. Despite its higher molar weight, irinotecan appeared to penetrate the 3D spheroid structure more efficiently than 5-fluorouracil. While 5-fluorouracil mainly caused a suppression of spheroid growth from the outside, irinotecan affected the entire spheroid and caused its originally compact structure to disintegrate. The acquired results highlight the need to screen cancer chemotherapeutics on 3D tumor models, as contrasting results can be obtained compared to standard 2D cell cultures.
Insights
Three-dimensional (3D) cell cultures better mimic tumors than 2D models for anticancer drug screening. Drug efficacy varied significantly, with irinotecan penetrating 3D spheroids better than 5-fluorouracil, highlighting the need for 3D models.
Area of Science:
- Biomedical Engineering
- Cancer Research
- Drug Discovery
Background:
- Three-dimensional (3D) cell cultures offer a more realistic model of tumor microenvironments compared to traditional 2D cultures.
- This improved mimicry is crucial for accurate biological and anticancer drug screening.
- Colon adenocarcinoma spheroids (HT-29 cell line) were used to investigate drug penetration and efficacy.
Purpose of the Study:
- To investigate the growth kinetics of colon adenocarcinoma spheroids in different micro-bioreactors.
- To compare the therapeutic efficacy of 5-fluorouracil and irinotecan hydrochloride in 2D and 3D cell culture models.
- To understand how drug penetration through 3D tumor spheroids affects chemotherapeutic efficacy.
Main Methods:
- Cultivation of colon adenocarcinoma spheroids (HT-29) in liquid marble micro-bioreactors and PDMS-coated well plates.
- Precise control of spheroid size using seed cell density and cultivation time.
- Assessment of drug efficacy (5-fluorouracil, irinotecan hydrochloride) using IC50 values in both 2D and 3D cultures.
Main Results:
- Spheroid size was precisely controlled by seed cell density and cultivation time.
- Drug efficacy varied significantly between 2D and 3D models; IC50 for 5-fluorouracil increased from 11.3 µM (2D) to 707.7 µM (3D).
- Irinotecan showed better penetration into 3D spheroids than 5-fluorouracil, with IC50 increasing from 24.9 µM (2D) to 77.8 µM (3D), causing spheroid disintegration.
Conclusions:
- 3D tumor models are essential for accurate anticancer drug screening due to differential drug penetration.
- The efficacy of chemotherapeutics like 5-fluorouracil and irinotecan can be significantly overestimated in 2D cultures.
- Results underscore the importance of utilizing 3D microtissues to predict in vivo drug response.
