Development and Validation of a Long-Term 3D Glioblastoma Cell Culture in Alginate Microfibers as a Novel Bio-Mimicking Model System for Preclinical Drug Testing

Abstract

Background: Various three-dimensional (3D) glioblastoma cell culture models have a limited duration of viability. Our aim was to develop a long-term 3D glioblastoma model, which is necessary for reliable drug response studies. Methods: Human U87 glioblastoma cells were cultured in alginate microfibers for 28 days. Cell growth, viability, morphology, and aggregation in 3D culture were monitored by fluorescent and confocal microscopy upon calcein-AM/propidium iodide (CAM/PI) staining every seven days. The glioblastoma 3D model was validated using temozolomide (TMZ) treatments 3 days in a row with a recovery period. Cell viability by MTT and resistance-related gene expression (MGMT and ABCB1) by qPCR were assessed after 28 days. The same TMZ treatment schedule was applied in 2D U87 cell culture for comparison purposes. Results: Within a long-term 3D model system in alginate fibers, U87 cells remained viable for up to 28 days. On day 7, cells formed visible aggregates oriented to the microfiber periphery. TMZ treatment reduced cell growth but increased drug resistance-related gene expression. The latter effect was more pronounced in 3D compared to 2D cell culture. Conclusion: Herein, we described a long-term glioblastoma 3D model system that could be particularly helpful for drug testing and treatment optimization.

On optimal temozolomide scheduling for slowly growing gliomas

“…Although this is certainly the way to go when CT is intended as a curative treatment, it is not obvious that it would be the best strategy when it is known that treatment can only control disease for a limited time. Intensification of TMZ delivery schemes has been studied for either newly diagnosed 31 or recurrent 32,33 high-grade gliomas without positive results on OS and with increased toxicity 38 . However, no previous studies have considered effective dose-reduction schemes with longer time spacing between treatments.…”

“…Alginate microfibers with cells were produced by extrusion as described earlier 32 . U251 cell lines were immobilized in alginate microfibers by the same procedure.…”

Controlled Fabrication of Bioactive Microtubes for Screening Anti-Tongue Squamous Cell Migration Drugs

“…Additionally, alginate is transparent and subsequent experiments can be visualized to ensure stability during injection. (Dragoj et al, 2021). The coaxial microfluidic device was designed and manufactured to precisely fabricate hydrogel microtubes, which was consisted of a coaxial needle and gel channels.…”

“…Now microtubes with alginate core-shell structure have been successfully fabricated with self-designed and fabricated microfluidic devices. Dragoj reported a long-term observation system for three-dimensional glioblastoma based on syringeextruded alginate fibers (Dragoj et al, 2021). Jorgensen demonstrated the capacity of fabricating hydrogel microtubes of coaxial needle devices (Jorgensen et al, 2021).…”

“…Traditional cell cultures in vitro are two-dimensional (2D) and are used to observe various biological behaviors of cells and mechanisms of cancer progression, as well as the drug discovery process. (Yamada and Sixt, 2019;Dragoj et al, 2021). However, 2D cell culture models in vitro fail to correctly imitate the threedimensional (3D) environment in vivo.…”