ABSTRACT

Purpose: The rotary cell culture system (RCCS) is a common clinorotation device for cell culture. It is also used as a low-shear suspension culture bioreactor to form functionalized 3D tissue constructs and to model microgravity. We sought to develop a 3D scaffold composed of type I collagen and hydroxyapatite (collagen-HA) to characterize MLO-Y4 osteocytes following suspension culture or clinorotation.

Materials and Methods: MLO-Y4 cells were embedded in collagen-HA. The scaffold was formed into droplets for suspension culture or wall-adhered to the RCCS for clinorotation. AFM, rheometry, immunofluorescence and qRT-PCR were employed to measure the scaffold stiffness, cell viability and gene expression of cells in collagen-HA scaffolds. Dendritic cells were visualized and quantified and gene expression after suspension culture and clinorotation was compared to static controls.

Results: The optimized scaffold for the RCCS consisted of collagen with 6 mg/mL HA which had a stiffness of < 1 kPa. MLO-Y4 cell viability was higher in collagen-HA scaffolds, compared to scaffolds without HA. Collagen-HA scaffolds induced higher osteocyte-specific gene expression compared to cells cultured on 2D plastic. Cells in the scaffold downregulated DMP1, E11, IL-6, and RANKL, and had fewer dendritic cells following suspension culture whereas clinorotation downregulated DMP1 and E11 genes, compared to static controls.

Conclusions: Suspension culture for 3 days in collagen-HA stimulates growth of osteocytes but may also desensitize them to mechanical cues. Clinorotation for 3 days in collagen-HA does not stimulate proliferation or expression of mechanosensitive genes, indicating that it may be an effective mechanical unloading environment.

摘要

用途： 旋转细胞培养系统 (RCCS) 是一种用于细胞培养的常用倾斜旋转装置。它还用作低剪切悬浮培养生物反应器，以形成功能化的 3D 组织结构并模拟微重力。我们试图开发一种由 I 型胶原蛋白和羟基磷灰石 (collagen-HA) 组成的 3D 支架，以表征悬浮培养或斜旋转后的 MLO-Y4 骨细胞。

材料和方法： MLO-Y4 细胞嵌入胶原蛋白-HA 中。支架形成液滴用于悬浮培养或壁粘附在 RCCS 上用于倾斜旋转。采用 AFM、流变仪、免疫荧光和 qRT-PCR 来测量胶原蛋白-HA 支架中细胞的支架刚度、细胞活力和基因表达。对树突状细胞进行可视化和量化，并将悬浮培养和倾斜旋转后的基因表达与静态对照进行比较。

结果：优化的 RCCS 支架由具有 6 mg/mL HA 的胶原蛋白组成，其刚度 < 1 kPa。与不含 HA 的支架相比，胶原蛋白-HA 支架中的 MLO-Y4 细胞活力更高。与在 2D 塑料上培养的细胞相比，胶原蛋白-HA 支架诱导更高的骨细胞特异性基因表达。与静态对照相比，支架中的细胞下调DMP1、E11、IL-6和RANKL，并且在悬浮培养后具有较少的树突状细胞，而旋转则下调DMP1和E11基因。

结论：在胶原蛋白-HA 中进行 3 天的悬浮培养可刺激骨细胞的生长，但也可能使它们对机械信号不敏感。在胶原蛋白-HA 中旋转 3 天不会刺激机械敏感基因的增殖或表达，表明它可能是一种有效的机械卸载环境。