Cell cryopreservation has evolved as an important technology required for supporting various cell-based applications, such as stem cell therapy, tissue engineering, and assisted reproduction. Recent times have witnessed an increase in the clinical demand of these applications, requiring urgent improvements in cell cryopreservation. However, cryopreservation technol-ogy suffers from the issues of low cryopreservation efficiency and cryoprotectant (CPA) toxicity. Application of advanced biotechnology tools can significantly improve post-thaw cell survival and reduce or even eliminate the use of organic solvent CPAs, thus promoting the development of cryopreservation. Herein, based on the different cryopreservation mechanisms available, we provide an overview of the applications and achievements of various biotechnology tools used in cell cryo-preservation, including trehalose delivery, hydrogel-based cell encapsulation technique, droplet-based cell printing, and nanowarming, and also discuss the associated challenges and perspectives for future development.