Reproducible 3D culture of multicellular tumor spheroids in supramolecular hydrogel from cancer stem cells sorted by sedimentation field-flow fractionation.

in Journal of chromatography. A by Tarek Saydé, Omar El Hamoui, Bruno Alies, Gaëlle Bégaud, Barbara Bessette, Sabrina Lacomme, Philippe Barthélémy, Gaëtane Lespes, Serge Battu, Karen Gaudin

TLDR

  • The study investigates the growth of cancer stem cells (CSCs) in a 3D environment. The CSCs were sorted using a specific method and then cultured in a hydrogel. The study found that the CSCs grown in the hydrogel formed spheroids with a consistent size, while the spheroids grown from unsorted cells were more variable in size. This suggests that the hydrogel provides a more reliable environment for growing CSCs in a 3D culture.

Abstract

Three-dimensional (3D) cancer models, such as multicellular tumor spheroids (MCTS), are biological supports used for research in oncology, drug development and nanotoxicity assays. However, due to various analytical and biological challenges, the main recurring problem faced when developing this type of 3D model is the lack of reproducibility. When using a 3D support to assess the effect of biologics, small molecules or nanoparticles, it is essential that the support remains constant over time and multiples productions. This constancy ensures that any effect observed following molecule exposure can be attributed to the molecule itself and not to the heterogeneous properties of the 3D support. In this study, we address these analytical challenges by evaluating for the first time the 3D culture of a sub-population of cancer stem cells (CSCs) from a glioblastoma cancer cell line (U87-MG), produced by a SdFFF (sedimentation field-flow fractionation) cell sorting, in a supramolecular hydrogel composed of single, well-defined molecule (bis-amide bola amphiphile 0.25% w/v) with a stiffness of 0.4 kPa. CSCs were chosen for their ability of self-renewal and multipotency that allow them to generate fully-grown tumors from a small number of cells. The results demonstrate that CSCs cultured in the hydrogel formed spheroids with a mean diameter of 336.67 ± 38.70 µm by Day 35, indicating reproducible growth kinetics. This uniformity is in contrast with spheroids derived from unsorted cells, which displayed a more heterogeneous growth pattern, with a mean diameter of 203.20 ± 102.93 µm by Day 35. Statistical analysis using an unpaired t-test with unequal variances confirmed that this difference in spheroid size is significant, with a p-value of 0.0417 (p < 0.05). These findings demonstrate that CSC-derived spheroids, when cultured in a well-defined hydrogel, exhibit highly reproducible growth patterns compared to spheroids derived from unsorted cells, making them a more reliable 3D model for biological research and drug testing applications.

Overview

  • The study evaluates the 3D culture of a sub-population of cancer stem cells (CSCs) from a glioblastoma cancer cell line (U87-MG), produced by a SdFFF (sedimentation field-flow fractionation) cell sorting, in a supramolecular hydrogel composed of single, well-defined molecule (bis-amide bola amphiphile 0.25% w/v) with a stiffness of 0.4 kPa. The primary objective of the study is to assess the reproducibility of CSC-derived spheroids when cultured in a well-defined hydrogel compared to spheroids derived from unsorted cells. The study aims to provide a reliable 3D model for biological research and drug testing applications.

Comparative Analysis & Findings

  • The study compares the growth kinetics of CSC-derived spheroids cultured in a well-defined hydrogel with spheroids derived from unsorted cells. The results demonstrate that CSC-derived spheroids exhibited highly reproducible growth patterns compared to spheroids derived from unsorted cells. The mean diameter of CSC-derived spheroids was 336.67 ± 38.70 µm by Day 35, while the mean diameter of spheroids derived from unsorted cells was 203.20 ± 102.93 µm by Day 35. Statistical analysis using an unpaired t-test with unequal variances confirmed that this difference in spheroid size is significant, with a p-value of 0.0417 (p < 0.05).

Implications and Future Directions

  • The study's findings demonstrate that CSC-derived spheroids, when cultured in a well-defined hydrogel, exhibit highly reproducible growth patterns compared to spheroids derived from unsorted cells. This makes them a more reliable 3D model for biological research and drug testing applications. Future research directions could include exploring the use of different hydrogel compositions and stiffnesses to optimize the growth and reproducibility of CSC-derived spheroids. Additionally, further studies could investigate the use of CSC-derived spheroids for drug testing and nanotoxicity assays.
Read Full Article