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Background: Adipose-derived stem cell (ADSC) sheets have been shown to promote tendon-to-bone healing. However, conventional laboratory preparation methods for ADSC sheets are time-consuming and risky, which precludes their diverse clinical applications. Purpose: To explore the utility of off-the-shelf cryopreserved ADSC sheets (c-ADSC sheets) for rotator cuff tendon-to-bone healing. Study Design: Controlled laboratory study. Methods: The ADSC sheets were cryopreserved and thawed for live/dead double staining, TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, scanning electron microscopy observation, and biomechanical testing. Clone formation, proliferative capacity, and multilineage differentiation of ADSCs within the c-ADSC sheets were assayed to explore the effect of cryopreservation on stem cell properties. A total of 67 rabbits were randomly divided into 4 groups: normal group (without supraspinatus tendon tears; n = 7), control group (repair alone; n = 20), fresh ADSC (f-ADSC) sheet group (repair; n = 20), and c-ADSC sheet group (repair; n = 20). Rabbit bilateral supraspinatus tendon tears were induced to establish a chronic rotator cuff tear model. Gross observation, micro–computed tomography analysis, histological or immunohistochemical tests, and biomechanical tests were conducted at 6 and 12 weeks after repair. Results: No significant impairment was seen in the cell viability, morphology, and mechanical properties of c-ADSC sheets when compared with f-ADSC sheets. The stem cell properties of ADSC sheets also were preserved by cryopreservation. At 6 and 12 weeks after the repair, the f-ADSC and c-ADSC sheet groups showed superior bone regeneration, higher histological scores, larger fibrocartilage areas, more mature collagen, and better biomechanical results compared with the control group. No obvious difference was seen between the f-ADSC and c-ADSC sheet groups in terms of bone regeneration, histological score, fibrocartilage formation, and biomechanical tests. Conclusion: c-ADSC sheets, an off-the-shelf scaffold with a high potential for clinical translational application, can effectively promote rotator cuff tendon-to-bone healing. Clinical Relevance: Programmed cryopreservation of ADSC sheets is an efficient off-the-shelf scaffold for rotator cuff tendon-to-bone healing. [ABSTRACT FROM AUTHOR]