Droplet printing to generate 3D tissue model

CELLINK Life Sciences is a world’s leading company in bioprinting research field and by combining advances in biology, software and engineering they also represent the top bio-convergence company in the world, creating the future of medicine.

At CELLINK, they develop innovative technologies to advance life science applications and, among them, the droplet tumor bioprinting allow to create droplets with one or more cell types and generate high-throughput 3D tissue models with high reproducibility for drug testing.

In particular, we adapted a 3D bioprinted tumor model where human pancreatic cancer cells (Panc1) and human umbilical vein endothelial cells (HUVECs) were mixed with 4% alginate, bioprinted using a Bio X and crosslined with calcium chloride. The 3D constructs were then cultured for a week before they were embedded in OCT media and kept frozen at -80C. Before fluorescent staining, the constructs were thawed on ice and washed thoroughly with PBS for few times. Alexa Fluor 647-conjugated phalloidin was used to detect cellular cytoskeleton (shown in magenta) and CFSE probes used to visualize the cells (shown in green). DAPI was used to detect nuclei structures (shown in blue). The droplets were kept in PBS 1X on glass-bottom dish while performing microscopy acquisitions.

We performed acquisitions with CrestOptics X-Light V3 spinning disk confocal coupled with LDI laser source (89 North) and sCMOS Prime BSI Camera (Photometrics, 6.5 um pixel size). We compared 20x air objective (0.7 NA and about 1 mm WD) and 30x silicone oil objective (1.05 NA and 0.8 mm WD). In Figure 1, we show maximum intensity projection (MIP) images obtained from Z stacks of about 140-280 um total thickness and acquired with 20x air objective suggesting complex cellular organization and density in the bioprinted droplet. To better appreciate the 3D cellular architecture, we show a volume view (300 um thickness) of one of the inner area shown in Figure 1 (bottom right) highlighting peculiar organization of cells mimicking different tissue layers (Figure 2). To gain in resolution, we moved to a 30x silicone oil objective with greater NA compare to the 20x and we report the MIP, the volume views and a 3D movie of a droplet outer area for a total thickness of 350 um (Figures 3 and 4).

Overall, due to the amount of CSFE positive cells revealed by confocal microscopy acquisitions, we can speculate that cell proliferation is more frequent in the external areas of the bioprinted droplet compared to the inner ones.

In basic and applied research, bioprinted tissue models offer the advantage to mimic the native phenotype of any cell type. They offer a great help in drug discovery field allowing researchers to understand the potential therapeutic effect of a compound in a physiological environment similar to human tissue. Lastly, bioprinted models enhance understanding of cellular maturation, communications, survival in both health conditions and disease models.

Figure 1: MIP of different droplet areas from Z stacks acquired with X-Light V3 confocal spinning disk and 20x air objective (TOP: outer areas; BOTTOM: inner areas)                                

Figure 2: Volume view of the inner area for a total thickness of 300 um.

Figure 3: MIP (TOP) and volume views (BOTTOM) of a droplet outer area from a Z stack acquired with X-Light V3 confocal spinning disk and 30x silicone oil objective for a total thickness of 350 um.  

Figure 4: 3D movie of the droplet area shown in Figure 3.

The application note has been prepared in collaboration with Shubhankar Nath, Himjyot Jaiswal and Itedale Namro Redwan from CELLINK LLC.

CELLINK Life Sciences 

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