Current slow-freezing methods are too inefficient for cryopreservation of three-dimensional (3D) tissue constructs. Additionally, conventional vitrification methods use liquid nitrogen, which is inconvenient and increases the chance of cross-contamination. Herein, we have developed polyampholytes with various degrees of hydrophobicity and showed that they could successfully vitrify cell constructs including spheroids and cell monolayers without using liquid nitrogen. The polyampholytes prevented ice crystallization during both cooling and warming, demonstrating their potential to prevent freezing-induced damage. Monolayers and spheroids vitrified in the presence of polyampholytes yielded high viabilities post-thawing with monolayers vitrified with PLL-DMGA exhibiting more than 90% viability. Moreover, spheroids vitrified in the presence of polyampholytes retained their fusibilities, thus revealing the propensity of these polyampholytes to stabilize 3D cell constructs. This study is expected to open new avenues for the development of off-the-shelf tissue engineering constructs that can be prepared and preserved until needed.