Spheroid formation

Spheroid formation of lung cancer cells

With new advances taking place frequently in cancer immunotherapy, gene editing and chemotherapy, there is a need for in vitro tumor models that can faithfully mimic the tumor environment for testing these new technologies and pharmaceutical drugs. An important step in evaluating more accurate in vitro tumor models are tumor spheroids which are multicellular aggregates with the property of dynamic cell-cell and cell-matrix interaction.


The aim of the following ongoing project is to study the possible spheroid formation of A549, a human lung adenocarcinoma cell line, after 3D bioprinting in various GelMA based of bioinks.



CELLINK Products Used

  • GelMA C
  • GelXG
  • BIO X

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.

3D Bioprinting to enable spheroid formation

In this project 3D bioprinting was used to investigate the spheroid formation of a lung cancer cell line in several of CELLINKs GelMA based bioinks. After mixing the A549 cells into GelMA C bioink, small droplets were printed in 96-well plates. The constructs were cultured for 14 days where visual observations of the cell aggregation were performed in a bright-field microscope.


Spheroid formation was detected after 14 days of culture which can be seen in the bright-field image beside. The image display three spheroids at the edge of the construct.

Aggregation and migration

Our GelXG bioink was used to culture A549 cells and study the cell viability and aggregation. The cells were mixed with the bioink and small droplets were printed and photocrosslinked. Within only 7 days of culture, clusters were observed that then grew larger up to day 14. At day 14 the cell viability was assessed which 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 size

A pure GelMA bioink was also used to print small droplets cell ladened with A549 lung cancer cells. The cells were mixed in at 5 million cells/mL of bioink and printed as droplets. The constructs were photocrosslinked and cultured up to 30 days. The cell aggregation could be observed in bright-field microscope due to the transparency of the GelMA bioink.


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


3D bioprinting of lung cancer cells in different GelMA based bioinks enables the cells to migrate, cluster together and form spheroids with a high viability. These 3D cancer models can be used to first study the migration of the cells and then on the formed spheroids, perform drug testing where the bioink can mimic the ECM surrounding the tumor in the body.