Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA.
Department of Cell and Molecular Biology, College of Environment and Life Sciences, University of Rhode Island, Kingston, RI 02881, USA.
Mater Sci Eng C Mater Biol Appl. 2021 Jun;125:112100. doi: 10.1016/j.msec.2021.112100. Epub 2021 Apr 14.
Complex three-dimensional (3D) cell cultures are being increasingly implemented in biomedical research as they provide important insights into complex cancer biology, and cell-cell and cell-matrix interactions in the tumor microenvironment. However, most methods used today for 3D cell culture are limited by high cost, the need for specialized skills, low throughput and the use of unnatural culture environments. We report the development of a unique biomimetic hydrogel microwell array platform for the generation and stress-free isolation of cancer spheroids. The poly N-isopropylacrylamide-based hydrogel microwell array (PHMA) has thermoresponsive properties allowing for the attachment and growth of cell aggregates/ spheroids at 37 °C, and their easy isolation at room temperature (RT). The reversible phase transition of the microwell arrays at 35 °C was confirmed visually and by differential scanning calorimetry. Swelling/ shrinking studies and EVOS imaging established that the microwell arrays are hydrophilic and swollen at temperatures <35 °C, while they shrink and are hydrophobic at temperatures >35 °C. Spheroid development within the PHMA was optimized for seeding density, incubation time and cell viability. Spheroids of A549, HeLa and MG-63 cancer cell lines, and human lung fibroblast (HLF) cell line generated within the PHMAs had relatively spherical morphology with hypoxic cores. Finally, using MG-63 cell spheroids as representative models, a proof-of-concept drug response study using doxorubicin hydrochloride was conducted. Overall, we demonstrate that the PHMAs are an innovative alternative to currently used 3D cell culture techniques, for the high-throughput generation of cell spheroids for disease modeling and drug screening applications.
复杂的三维（3D）细胞培养越来越多地应用于生物医学研究，因为它们为复杂的癌症生物学以及肿瘤微环境中的细胞-细胞和细胞-基质相互作用提供了重要的见解。然而，目前用于 3D 细胞培养的大多数方法都受到高成本、专业技能需求、低通量和非自然培养环境的限制。我们报告了一种独特的仿生水凝胶微井阵列平台的开发，用于生成和无应力分离癌症球体。基于聚 N-异丙基丙烯酰胺的水凝胶微井阵列（PHMA）具有温度响应特性，允许细胞聚集体/球体在 37°C 下附着和生长，并在室温（RT）下轻松分离。微井阵列在 35°C 的可逆相转变通过肉眼观察和差示扫描量热法得到证实。溶胀/收缩研究和 EVOS 成像表明，微井阵列在温度<35°C 时亲水且溶胀，而在温度>35°C 时收缩且疏水。在 PHMA 中优化了球体发育的接种密度、孵育时间和细胞活力。在 PHMAs 中生成的 A549、HeLa 和 MG-63 癌细胞系和人肺成纤维细胞（HLF）球体具有相对球形的形态，并有缺氧核心。最后，使用 MG-63 细胞球体作为代表性模型，进行了使用盐酸多柔比星的药物反应研究的概念验证。总的来说，我们证明 PHMAs 是目前使用的 3D 细胞培养技术的创新替代方法，可用于高通量生成用于疾病建模和药物筛选应用的细胞球体。
