| Summary: | For salivary gland (SG) tissue engineering, we cultured acinar NS-SV-AC cell line or primary SG fibroblasts for 14 days in avian egg yolk plasma (EYP). Media or egg white (EW) supplemented the cultures as they grew in 3D-Cryo histology well inserts. In the second half of this manuscript, we measured EYP’s freeze-thaw gelation and freeze-thaw induced gelled EYP (<sub>G</sub>EYP), and designed and tested further <sub>G</sub>EYP tissue engineering applications. With a 3D-Cryo well insert, we tested <sub>G</sub>EYP as a structural support for 3D cell culture or as a bio-ink for 3D-Bioprinting fluorescent cells. In non-printed EYP + EW or <sub>G</sub>EYP + EW cultures, sagittal sections of the cultures showed cells remaining above the well’s base. Ki-67 expression was lacking for fibroblasts, contrasting NS-SV-AC’s constant expression. Rheological viscoelastic measurements of <sub>G</sub>EYP at 37 °C on seven different freezing periods showed constant increase from 0 in mean storage and loss moduli, to 320 Pa and 120 Pa, respectively, after 30 days. We successfully 3D-printed <sub>G</sub>EYP with controlled geometries. We manually extruded <sub>G</sub>EYP bio-ink with fluorescence cells into a 3D-Cryo well insert and showed cell positioning. The 3D-Cryo well inserts reveal information on cells in EYP and we demonstrated <sub>G</sub>EYP cell culture and 3D-printing applications.
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