Cryopreservation of 3D Bioprinted Scaffolds with Temperature-Controlled-Cryoprinting

Abstract

Temperature-Controlled-Cryoprinting (TCC) is a new 3D bioprinting technology that allows for the fabrication and cryopreservation of complex and large cell-laden scaffolds. During TCC, bioink is deposited on a freezing plate that descends further into a cooling bath, keeping the temperature at the nozzle constant. To demonstrate the effectiveness of TCC, we used it to fabricate and cryopreserve cell-laden 3D alginate-based scaffolds with high cell viability and no size limitations. Our results show that Vero cells in a 3D TCC bioprinted scaffold can survive cryopreservation with a viability of 71%, and cell viability does not decrease as higher layers are printed. In contrast, previous methods had either low cell viability or decreasing efficacy for tall or thick scaffolds. We used an optimal temperature profile for freezing during 3D printing using the two-step interrupted cryopreservation method and evaluated drops in cell viability during the various stages of TCC. Our findings suggest that TCC has significant potential for advancing 3D cell culture and tissue engineering.

Keywords: 3D bioprinting; 3D cell culture; alginate; cryopreservation; freezing.

Figure 1

(a) Static cryoprinting versus Temperature-Controlled Cryoprinting. (b) A supply chain of cell-laden scaffolds from fabrication to use.

Figure 2

Multi-layer scaffolds printed with temperature-controlled cryoprinting. (a) Images taken with a thermal camera during printing demonstrate that the temperature distribution at the nozzle is constant at the first layer and higher layers. (b) An eight-layer line and an eight-layer hollow square printed with temperature-controlled cryoprinting.

Figure 3

(a) An optimal temperature profile for temperature-controlled cryoprinting and subsequent cooling to −80 °C. (b) Initial bioink temperature and extrusion onto a −5 °C print plate during temperature-controlled cryoprinting. (c) Cell viability rates versus initial bioink temperature. * p < 0.05, and error bars represent ± one standard deviation from the mean.

Figure 4

A comparison of cell viability in the first and fifth layers of five-layer scaffolds printed at −5 °C and cryopreserved at −80 °C. Images were taken at the left, middle, and right, along each layer. Fluorescence images include all cells (blue) and dead cells (red). The p-value was 0.963 and error bars represent ± one standard deviation from the mean.

Figure 5

Assessing cell viability during the different stages of the temperature-controlled cryoprinting process. The * p < 0.05, ** p < 0.01, and error bars represent ± one standard deviation from the mean.