The other week we challenged ourselves to fabricate perfusable networks within a broader hydrogel medium using the INKREDIBLE+. We aimed to make the procedure as simple as possible to allow rapid prototyping of network design. While the steps outline below were performed to fabricate one network architecture within one type of hydrogel medium, the approach is adaptable. One can imagine fabricating these networks to engineer hypoxic tumor environments or drug delivery to diseased cells, cell migration studies, or the influence of diffusible biochemical cues inconjugation with matrix cues such as stiffness or density, etc. The possibilities are endless!

 

Follow up blog posts will illustrate the ability to deposit specific materials (and cells) in specific regions that are perfused by the network. As always we hope to make these protocols and gcode files available to you the researcher, they will be up on Bioverse in the coming weeks. As always, if you have any questions you can contact Dr. Patrick Thayer at pt ‘at’ cellink.com.

 

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Figure 1: Bioprinting of Pluronics-based channels on a standard microscope slide

 

We first aimed to restrict our network at the time to a standard (75 mm by 25 mm) microscope slide (Figure 1).  We decided that a network that branches a single time is boring, so we decided to print a network that branches twice, then returns to a single outlet. Additionally, each time the vessel branches, its diameter is decreased. You can see this in Figure 2A, where level 0 of the network consists of one vessel which then splits into two vessels at level 1 of the network (each with a smaller diameter), this continues at the split to level 2 of the network which consists of 4 vessels each with smaller diameters than the previous level. For this first test of the network we cast gelatin over the printed network to encapsulate it. Then cooled the slide and displaced the pluronics with a red dye to illustrate the perfusability of the printed network (Figure 2B).

 

Figure 2: A– bioprinted network encapsulated in gelatin, network branching visible, vessel diameter decreases with increasing degree of branching. B– Perfused network through all branches with a red dye.

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The printed network show in Figure 2B was a good start. However, the major limitation was that the network had to be encapsulated after the printing process manually. It was difficult to evenly distribute the gelatin over the printed network, the resulting gelatin layer was irregular in places and often resulted in incorporation of airbubbles that disrupted perfusion. To address this, the gcode was modified to allow the encapsulation of the printed network during the bioprinting process. As shown in Figure 3A, a dual cartridge set-up was utilized. The first cartridge was CELLINK GelMA and was heated slighted to result in a more fluid bioink compared to the normal viscosity of GelMA. The second cartridge was CELLINK Pluronics, this time containing a dye for better visualization of the network prior to perfusion. The printbed was heated to 37C to both retain the shape of the Pluronics after printing but to also liquidify the GelMA after printing to allow it to completely encapsulate the Pluronics network.

 

As shown in the video . The network was printed utilizing the Pluronics onto a standard microscope slide (~30 seconds total). Then the GelMA was printed on either side of the Pluronic filaments. As you can see the GelMA began to liquidify and spread to fill in the gaps. Once the GelMA was spread (~ another 30 seconds total). The temperature of the print bed was dropped to 15 C to begin to solidify the GelMA. After solidification, the GelMA was cross-linked under UV. The final encapsulated network is shown in Figure 3B.

 

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Figure 3: A– dual cartridge printing, Cartridge 1 -> CELLINK GelMA, Cartridge 2 -> CELLINK Pluronics, B– Printed network encapsulated in Gelatin methacrylate

 

After cross-linking, the encapsulated network was placed on ice to liquidify the pluronics, then perfused with dyed water. Can see the before perfusion (Figure 4A) and after perfusion (Figure 4B), printed pluronic network on bottom for reference.

 

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Figure 4: A – Before Perfusion, B – After Perfusion

 

 

Thanks for reading! Stay tuned for more blog posts in the future about more complicated networks that are both symmetrical and random (including the CELLINK Perfusable Network Gcode Generator!), a new bioink kit that will provide all the tools you need to rapidly prototype perfusable networks, and other cool stuff that you can do with a bioprinter! As always contact Dr. Patrick Thayer if you have any questions or want to show off the cool stuff that you print!

 

 

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