3D tumor spheroids on Nunclon Sphera 3D cell culture plates

Nunclon Sphera 3D culture system

Thermo Scientific Nunclon Sphera culture system is designed for spheroid culture (e.g., 3D tumor spheroids) and organoid culture (e.g., brain organoids) with a wide selection of format from plates to dishes and flasks. The 3D cell culture surface allows virtually no cell attachment to the cultureware, supporting the establishment of 3D spheroids and organoids via cell-to-cell aggregation through naturally secreted extracellular matrices (ECM).

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Low cell attachment surface

Proprietary surface coating of Nunclon Sphera prohibits any protein adsorption to the cultureware surface, thereby minimizing monolayer cell adhesion to the culture vessel.

Bar chart showing reduced attachment for Nunclon Sphera surface

Figure 1. Compared to the standard cell culture flask (e.g., Nunclon Delta), minimal cell attachment was observed on the Nunclon Sphera 3D culture flask across 3 different cell types.

Consistent 3D tumor spheroid formation

The reliable cell growth and the ease in controlling the spheroid size on the Nunclon Sphera 96-well U-bottom 3D cell culture plate are important for applications where consistency and reproducibility are valued. 3D tumor spheroids imitate the in vivo development of solid tumors with homogenous growth and, in the cases of sizable tumor, the hypoxic core.

Microscopic views of early HCT 116 3D tumor spheroids cultured on Nunclon Sphera surface

Figure 2. Growth kinetics of HCT 116 colon cancer cells shows early spheroid formation in the Nunclon Sphera 3D cell culture plate, even with low initial seeding density of 100 cells/well.

Microscopic views of HCT 116 3D tumor spheroids at different seeding densities on Nunclon Sphera surface

Figure 3. Size of the HCT 116 3D tumor spheroids can be controlled by the initial seeding densities in the wells. HCT 116 colon cancer cells were cultured on the Nunclon Sphera 3D cell culture plate for 112 hours.
 

Microscopic view of hypoxic core of HeLa 3D tumor spheroid cultured on Nunclon Sphera surface

Figure 4. The hypoxic core in a HeLa tumor spheroid after 2 days of culture on the Nunclon Sphera 96-well U-bottom 3D cell culture plate with initial seeding density of 1000 cells/well. The 3D tumor spheroid was stained with Invitrogen Image-iT Red Hypoxia Probe and Invitrogen NucBlue Live ReadyProbe reagent on a Nunc glass bottom dish.

3D cell culture applications

Neurosphere and brain organoid generation

Through endogenously secreted natural ECM, neural stem cells (NSC) aggregate with one another, leading to rapid formation of neurosphere. With specific protocols, consistent cell growth in neurosphere can lead to further differentiation to multi-cellular structures known as brain organoid with tissue-specific morphology and function.

Microscopic views of neurospheres growing on Nunclon Sphera surface

Figure 5. Human iPSC-derived neurospheres were imaged and stained with DAPI after being cultured on the Nunclon Sphera 3D cell culture plate for a week.

Microscopic view of brain organoid cultured on Nunclon Sphera surface

Figure 6. Brain organoid cultured in the Nunclon Sphera 3D cell culture dish was immunostained and imaged using Invitrogen EVOS FL Auto 2 imaging system at 10x magnification.

Embryoid body formation

Embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) form 3D aggregates, known as embryoid body (EB), held together by complex network of adhesion molecules. The paracrine signaling within an embryoid body is crucial to embryogenesis in respond to environmental cues that lead to the eventual differentiation of three germ layers.

Microscopic view of viability of embryoid body cultured on Nunclon Sphera surface
Figure 7. Viability of human ESC in an EB cultured on the Nunclon Sphera 3D cell culture plate in Gibco Essential 6 medium supplemented with TGF-b is evaluated by Invitrogen LIVE (green)/DEAD (red) viability assay (scale bar 1000 μm).
Microscopic view of fluorescently stained germ layers in human embryoid bodies cultured on Nunclon Sphera surface

Figure 8. The differentiation of a human EB to three germ layers on Nunc Lab Tek II Chamber Slides as shown by  the expression of ectoderm marker beta-tubulin, endoderm marker alpha fetoprotein (AFP), and mesoderm marker smooth muscle actin (SMA). Counter-stained with DAPI.

Model system for high-throughput screening (HTS) in drug discovery

High failure rate of new drug development that incurs enormous cost for both pharmaceutic companies and consumers calls for better 3D cell models in the early stages of the drug development process. 3D cell spheroids and organoids, with uniform size and centered location in the wells of the Nunclon Sphera 3D cell culture microplates, present an attractive and more biologically relevant 3D cell model system for drug discovery and disease modeling where high-throughput screening on high-content screening platforms is often employed.

Microscopic view of A549 3D tumor spheroids in each well of 96-well Nunclon Sphera plate

Figure 9. A brightfield image of consistent A549 human lung 3D tumor spheroids with uniform size and shape in the Nunclon Sphera 96-well U-bottom plate.

Microscopic view of fluorescently stained A549 and HeLa 3D tumor spheroids in 96-well Nunclon Sphera plate

Figure 10. Niclosamide-induced mitochondrial membrane depolarization and apoptosis are demonstrated using A549 and HeLa 3D tumor spheroids on the Nunclon Sphera 96-well U-bottom plate. Tumor spheroids are stained with MitoTracker Orange and CellEvent Green Caspase 3/7.

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