Three-dimensional cultured patient-derived cancer organoids (PDOs) represent a powerful tool for anti-cancer drug development due to their similarity to the in vivo tumor tissues. However, the culture and manipulation of PDOs is more difficult than 2D cultured cell lines due to the presence of the culture matrix and the 3D feature of the organoids. In our other study, we established a method for lung cancer organoid (LCO)-based drug sensitivity tests on the superhydrophobic microwell array chip (SMAR-chip). Here, we describe a novel in situ cryopreservation technology on the SMAR-chip to preserve the viability of the organoids for future drug sensitivity tests. We compared two cryopreservation approaches (slow freezing and vitrification) and demonstrated that vitrification performed better at preserving the viability of LCOs. Next, we developed a simple procedure for in situ cryopreservation and thawing of the LCOs on the SMAR-chip. We proved that the on-chip cryopreserved organoids can be recovered successfully and, more importantly, showing similar responses to anti-cancer drugs as the unfrozen controls. This in situ vitrification technology eliminated the harvesting and centrifugation steps in conventional cryopreservation, making the whole freeze–thaw process easier to perform and the preserved LCOs ready to be used for the subsequent drug sensitivity test.

Drug resistance remains a major obstacle in cancer therapy, and direct mechanical disruption of cellular structures offers a promising strategy for overcoming it, highlighting the urgent clinical need for an effective and universal pan‐cancer treatment. Here, a cryo‐responsive liquid metal (LM) nanotransformer platform is developed. It undergoes dramatic deformation under freezing, physically disrupting lysosomal membranes and inducing potent cytotoxicity, particularly against drug‐resistant tumors, thus offering a broadly applicable pan‐cancer treatment. To enhance deformation efficiency of LM transformers, bismuth (Bi) is incorporated into gallium (Ga) to form biphasic Bi‐Ga alloy particles, which reduces Ga supercooling and promotes crystallization during freezing. The biosafety of LM particles is evaluated across multiple organoid models upon maximum contact. Based on 11 tumor organoid models including lung, colorectal, and ovarian cancers, the significant anti‐tumor effects of LM‐assisted cryotherapy are demonstrated, particularly Bi‐Ga combined with chemotherapy illustrating the strongest anti‐tumor efficacy. Transcriptomic analysis reveals LM anti‐tumor mechanism on multiple cancers with altering gene expression related to necroptosis, metabolic regulation, and cellular stress. In addition, tumors receiving LM cryo‐treatment exhibited hallmark features of immunogenic cell death. In immune‐competent drug‐resistant lung organoids, Bi‐Ga cryo‐treatment modulates tumor microenvironment and enhances immunogenicity. Collectively, this study establishes a universal LM nanotransformer‐enabled pan‐cancer strategy, offering a clinically viable and drug‐free approach for overcoming drug resistance.