Tolentino M A Kristine, Seyedzadeh Mir Hadi, Peres Newton Gil, Du Eric Yiwei, Zhu Lin, Gaus Katharina, Goyette Jesse, Gooding J Justin
School of Chemistry and Australian Centre of NanoMedicine, University of New South Wales, Sydney, New South Wales, Australia.
European Molecular Biology Laboratory Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, New South Wales, Australia.
J Biomed Mater Res A. 2025 Jan;113(1):e37811. doi: 10.1002/jbm.a.37811. Epub 2024 Oct 21.
Three-dimensional (3D) in vitro models enable us to understand cell behavior that is a better reflection of what occurs in vivo than 2D in vitro models. As a result, developing 3D models for extracellular matrix (ECM) has been growing exponentially. Most of the efforts for these 3D models are geared toward understanding cancer cells. An intricate network of cells that engages with cancer cells and can kill them are the immune cells, particularly cytotoxic T lymphocytes (CTLs). However, limited reports are available for 3D ECM mimics to understand CTL dynamics. Currently, we lack ECM mimetic hydrogels for immune cells, with sufficient control over variables, such as stiffness, to fully understand CTL dynamics in vitro. Here, we developed PEG-based hydrogels as ECM mimics for CTLs. The ECM mimics are targeted to mimic the stiffness of soft tissues where CTLs reside, migrate, and deliver their function. To understand cell-material interaction, we determined the porosity, biocompatibility, and stiffness of the ECM mimics. The ECM mimics have median pore sizes of 10.7 and 13.3 μm, close to the average nucleus size of CTLs (~8.6 μm), and good biocompatibility to facilitate cell migration. The stiffness of the ECM mimics corresponds to biologically relevant microenvironments such as lungs and kidneys. Using time-lapse fluorescence microscopy, 3D cell migration was imaged and measured. CTLs migrated faster in softer ECM mimic with larger pores, consistent with previous studies in collagen (the gold standard ECM mimic for CTLs). The work herein demonstrates that the PEG-based ECM mimic can serve as an in vitro tool to elucidate the cell dynamics of CTLs. Thus, this study opens possibilities to study the mechanics of CTLs in well-defined ECM mimic conditions in vitro.
三维（3D）体外模型使我们能够了解细胞行为，相较于二维体外模型，它能更好地反映体内发生的情况。因此，用于细胞外基质（ECM）的3D模型的开发呈指数级增长。这些3D模型的大部分工作都致力于了解癌细胞。与癌细胞相互作用并能杀死癌细胞的复杂细胞网络是免疫细胞，尤其是细胞毒性T淋巴细胞（CTLs）。然而，关于用于理解CTL动态的3D ECM模拟物的报道有限。目前，我们缺乏用于免疫细胞的ECM模拟水凝胶，无法对诸如硬度等变量进行充分控制，以全面了解体外CTL的动态。在此，我们开发了基于聚乙二醇（PEG）的水凝胶作为CTL的ECM模拟物。这些ECM模拟物旨在模拟CTL驻留、迁移和发挥功能的软组织的硬度。为了了解细胞与材料的相互作用，我们测定了ECM模拟物的孔隙率、生物相容性和硬度。ECM模拟物中位孔径分别为10.7和13.3μm，接近CTL的平均细胞核大小（约8.6μm），并且具有良好的生物相容性以促进细胞迁移。ECM模拟物的硬度与肺和肾等生物学相关的微环境相对应。使用延时荧光显微镜对3D细胞迁移进行成像和测量。CTL在具有较大孔隙的较软ECM模拟物中迁移得更快，这与先前在胶原蛋白（CTL的金标准ECM模拟物）中的研究一致。本文的工作表明，基于PEG的ECM模拟物可作为一种体外工具来阐明CTL的细胞动态。因此，本研究为在体外明确的ECM模拟条件下研究CTL的力学机制开辟了可能性。
