Bioprinting of vascular GBM model

The bioprinted model was fabricated as described in (1-2). A layer of collagen precursor was printed, then vaporized NaHCO₃ was applied to polymerize the collagen. A commercial humidifier (SPT) was used to vaporize sterile 0.8M NaHCO₃ solution. This process was repeated for 4-6 times to prepare ~1mm of collagen base. After then, 10% gelatin heated at 37°C was printed in two straight lines and allowed to be solidified at room temperature for ~1 minute. [Fig. 1, bioprinting (steps 1 and 2)]. Then, a GBM spheroid was picked using a pipette and placed in between the two gelatin channels [Fig. 1, bioprinting (step 3)]. Excessive medium around the spheroid was carefully removed using a pipette, and a small amount of collagen precursor was added to fix the spheroid location. More collagen layers were printed on top of the gelatin channels and the GBM spheroid. NaHCO₃ vapor was applied for every collagen layer printing [Fig. 1, bioprinting (step 4)]. The whole structure was then incubated at 37°C for 20 to 30 min to liquefy gelatin and obtain fluidic channels. Warm DPBS was injected into the printed channel to wash out the gelatin. After 3-4 times of DPBS washing, HUVECs in suspension were injected into the channels (seeding density: 8 million cells/ml), incubated for ~1 hour, then new culture medium was injected to clear out non-attached cells. The attached HUVECs created cell lining on the inner channel surface [Fig. 1, bioprinting (step 5)]. The entire construct was printed in a flow chamber, which allows stable, long-term perfusion (Fig. 1). The construct was cultured with EGM-2 medium for up to 70 days at 37°C in 5% CO₂. The culture medium was changed three to four times a day through the vascular channels.

1.     V. K. Lee, A. M. Lanzi, H. Ngo, S.-S. Yoo, P. A. Vincent, G. Dai, Generation of multi-scale vascular network system within 3D hydrogel using 3D bio-printing technology. Cell. Mol. Bioeng. 7, 460–472 (2014).

2.     V. K. Lee, D. Y. Kim, H. Ngo, Y. Lee, L. Seo, S.-S. Yoo, P. A. Vincent, G. Dai, Creating perfused functional vascular channels using 3D bio-printing technology. Biomaterials 35, 8092–8102 (2014).

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