Spheroid formation

Spheroid formation of lung cancer cells
Recent advances in cancer immunotherapy, gene editing and chemotherapy underscore the need for in vitro tumor models that can reliably test new technologies and pharmaceutical drugs. Tumor spheroids are multicellular aggregates with dynamic cell-cell and cell-matrix interaction properties, and they play an important role in developing more accurate in vitro tumor models.


The aim of our project is to study the spheroid formation of A549, a human lung adenocarcinoma cell line, after bioprinting in our GelMA-based bioinks.

Spheroid formation

Spheroids are multicellular cell aggregates that form via ECM fibres that link singles cells together through integrin binding. The cell-cell contact lead to an increased E-cadherin expression and these E-cadherin interactions generated the formation of the compact structures. Spheroids are more complex then single cells due to dynamic cell-cell and cell-matrix interaction which makes them an important tool for resembling the in vivo tissues microenvironment in vitro.


The multiphoton image beside show spheroids formed in GelXG bioink after 14 days of culture.

Bioprinting enables spheroid formation

At CELLINK, we used bioprinting to investigate the spheroid formation of a lung cancer cell line in our GelMA-based bioinks. After mixing the A549 cells into GelMA C bioink, we printed small droplets in 96-well plates. The constructs were cultured for 14 days and observed cell aggregation using a brightfield microscope.


Spheroid formation was detected after 14 days of culture, shown in the brightfield image to the right. The image displays three spheroids at the edge of the construct.

Aggregation and migration

We used GelXG bioink to culture A549 cells and study cell viability and aggregation. We mixed the bioink with cells before printing and photocuring small droplets. Within only seven days of culture, clusters formed and continued growing up to day 14. At day 14, cell viability was over 85%.


The 3D bioprinted cancer model demonstrates that the cells can form aggregates and migrate freely within the photocured GelXG. The multiphoton image beside, taken at day 14, show a cancer cell cluster (yellow) and tunnels, demonstrating proof of migration within the bioink (purple).

Spheroids of different sizes

We also used a pure GelMA bioink to print small droplets of cells laden with A549 lung cancer cells. We mixed cells in at five million cell per mL of bioink and printed them as droplets. The constructs were photocured and cultured for 30 days. The transparency of the GelMA bioink enabled us to observe cell aggregation using a brightfield microscope .


At the time of printing, the cells were homogeneously distributed in the bioink. Within 16 days of culture, the cells migrated and clustered. Both small clusters and larger spheroids up to 150 µm in diameter formed.


Bioprinting lung cancer cells in CELLINK’s GelMA-based bioinks enables the cells to migrate, cluster together and form spheroids with a high cell viability. These 3D cancer models can be used to study cell migration and test drugs on the spheroids formed.

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