Grenier Jérôme, David Bertrand, Journé Clément, Cicha Iwona, Letourneur Didier, Duval Hervé
Laboratoire de Génie des Procédés et Matériaux, CentraleSupélec, Université Paris-Saclay, 91190 Gif-sur-Yvette, France.
Laboratoire de Mécanique Paris-Saclay, CNRS, CentraleSupélec, ENS Paris-Saclay, Université Paris-Saclay, 91190 Gif-sur-Yvette, France.
Bioengineering (Basel). 2023 Jul 18;10(7):849. doi: 10.3390/bioengineering10070849.
The traditional 3D culture systems in vitro lack the biological and mechanical spatiotemporal stimuli characteristic to native tissue development. In our study, we combined porous polysaccharide-based hydrogel scaffolds with a bioreactor-type perfusion device that generates favorable mechanical stresses while enhancing nutrient transfers. MC3T3E1 mouse osteoblasts were seeded in the scaffolds and cultivated for 3 weeks under dynamic conditions at a perfusion rate of 10 mL min. The spatial distribution of the cells labeled with superparamagnetic iron oxide nanoparticles was visualized by MRI. Confocal microscopy was used to assess cell numbers, their distribution inside the scaffolds, cell viability, and proliferation. The oxygen diffusion coefficient in the hydrogel was measured experimentally. Numerical simulations of the flow and oxygen transport within the bioreactor were performed using a lattice Boltzmann method with a two-relaxation time scheme. Last, the influence of cell density and spheroid size on cell oxygenation was investigated. The cells spontaneously organized into spheroids with a diameter of 30-100 μm. Cell viability remained unchanged under dynamic conditions but decreased under static culture. The cell proliferation (Ki67 expression) in spheroids was not observed. The flow simulation showed that the local fluid velocity reached 27 mm s at the height where the cross-sectional area of the flow was the smallest. The shear stress exerted by the fluid on the scaffolds may locally rise to 100 mPa, compared with the average value of 25 mPa. The oxygen diffusion coefficient in the hydrogel was 1.6×10-9 m s. The simulation of oxygen transport and consumption confirmed that the cells in spheroids did not suffer from hypoxia when the bioreactor was perfused at 10 mL min, and suggested the existence of optimal spheroid size and spacing for appropriate oxygenation. Collectively, these findings enabled us to define the optimal conditions inside the bioreactor for an efficient in vitro cell organization and survival in spheroids, which are paramount to future applications with organoids.
传统的体外3D培养系统缺乏天然组织发育所特有的生物和机械时空刺激。在我们的研究中，我们将基于多孔多糖的水凝胶支架与生物反应器型灌注装置相结合，该装置在增强营养物质传递的同时产生有利的机械应力。将MC3T3E1小鼠成骨细胞接种到支架中，并在动态条件下以10 mL/min的灌注速率培养3周。通过MRI可视化超顺磁性氧化铁纳米颗粒标记细胞的空间分布。共聚焦显微镜用于评估细胞数量、它们在支架内的分布、细胞活力和增殖情况。通过实验测量水凝胶中的氧扩散系数。使用具有双弛豫时间格式的格子玻尔兹曼方法对生物反应器内的流动和氧传输进行了数值模拟。最后，研究了细胞密度和球体大小对细胞氧合的影响。细胞自发组织成直径为30-100μm的球体。细胞活力在动态条件下保持不变，但在静态培养下降低。未观察到球体中的细胞增殖（Ki67表达）。流动模拟表明，在流动横截面积最小的高度处，局部流体速度达到27 mm/s。与25 mPa的平均值相比，流体对支架施加的剪切应力可能局部升至100 mPa。水凝胶中的氧扩散系数为1.6×10-9 m²/s。氧传输和消耗的模拟证实，当生物反应器以10 mL/min的速率灌注时，球体中的细胞不会缺氧，并表明存在最佳的球体大小和间距以实现适当的氧合。总的来说，这些发现使我们能够确定生物反应器内的最佳条件，以实现体外细胞的有效组织和球体中的存活，这对于类器官的未来应用至关重要。
