Huizhong Hao,Xiuqiang Li,Chaojie Yu,Rui Liu,Jianying Hao,Xiang Ji,Qingyu Yu,Dunwan Zhu,Junjie Li
During two-dimensional (2D) culture, stem cells gradually lose their proliferative activity and multipotency due to various physicochemical conditions, which significantly hinder the large-scale clinical application of stem cell therapy. In recent years, three-dimensional (3D) cell culture has been increasingly utilized in the field of stem cell expansion owing to its unique advantages. The superhydrophilicity of zwitterionic hydrogels ensures the maintenance of stem cell stemness during their expansion. This study aims to address a key challenge in the large-scale culture of stem cells in vitro: how to sustain their proliferative capacity and multipotency while achieving efficient cell recovery. To this end, we have designed a novel zwitterionic degradable hydrogel based on host-guest interactions as a 3D carrier for the in vitro culture of adipose-derived stem cells (ADSCs). We synthesized the copolymer poly(sulfobetaine-co-cyclodextrin) (P(SBMA-co-CD)) and adamantane-grafted hyaluronic acid (HA-Ada), and a stable hydrogel was rapidly formed by simply mixing solutions of these two polymers. Leveraging the antifouling properties of zwitterionic groups, this hydrogel effectively maintained the long-term stemness expression of ADSCs during culture. More importantly, we utilized the reversibility of host-guest interactions to disrupt the cross-linked structure of the hydrogel by adding competitive monomers, enabling efficient recovery of stem cells under gentle conditions. This process not only achieved a high recovery rate of stem cells but also avoided the damage to cells caused by traditional cell recovery methods. In summary, this study creatively introduced host-guest interactions into zwitterionic hydrogels and successfully applied it to the 3D culture and recovery of stem cells in vitro. This hydrogel demonstrates functional plasticity in stem cell proliferation, culture, and harvest, holding promise for providing more reliable and efficient solutions in the fields of stem cell therapy and tissue engineering.
Background Lung cancer is one of the most common malignant tumors worldwide. Despite advances in lung cancer treatment, patients still face challenges related to drug resistance and recurrence. Current methods for evaluating anti-cancer drug activity are insufficient, as they rely on two-dimensional (2D) cell culture and animal models. Therefore, the development of an in vitro drug evaluation model capable of predicting individual sensitivity to anti-cancer drugs would greatly enhance the success rate of drug treatments for lung cancer patients. The purpose of this research is to utilise conditional reprogramming technology to cultivate patient-derived lung cancer cells and to construct an in vitro 3D culture model using sodium alginate (SA) and gelatin. The aim is to study the biological characteristics of cells in the 3D culture model and to further investigate the sensitivity of anti-cancer drugs based on the alginate-gelatin 3D culture model. This approach provides new means and insights for personalized precision anti-cancer therapy and the development of new anti-cancer drugs. Methods Conditional reprogramming technology was used to generate conditionally reprogrammed lung adenocarcinoma cells (CRLCs). Alginate-gelatin hydrogel micro-beads were created to explore their potential use in the assessment of anti-cancer drugs. Cell proliferation was also examined using the MTS assay method. Live/dead staining was performed to estimate cell distribution and viability using calcein acetoxymethyl ester/propidium iodide (calcein-AM/PI) double staining. Protein expression was assessed by Western blot. Results The cells grown in the three-dimensional (3D) culture were in a state of continuous proliferation, and there was an obvious phenomenon of cell mass growth. The drug sensitivity assay results demonstrated that compared with the 2D-grown cells, the CRLCs grown in the alginate-gelatin hydrogel micro-beads exhibited more resistance to anti-cancer drugs. The results also showed that the 3D-cultured CRLCs showed greater protein expression levels of stem cell hallmarks, such as Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX-2), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1), than the 2D-grown cells. Conclusions These findings suggest that the 3D hydrogel cell culture models more closely mimicked the in vivo biological and clinical behavior of cells, and demonstrated higher innate resistance to anti-cancer drugs than the 2D cell culture models, and thus could serve as valuable tools for diagnosis, drug screening, and personalized medicine.
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