Three-Dimensional Flow Perfusion Culture System for Stem Cell Proliferation Inside the Critical-Size β-Tricalcium Phosphate Scaffold

Abstract

A 3-dimensional flow perfusion system has been created in our laboratory to provide continuous and homogeneous nutrient supply inside the critical-size beta-tricalcium phosphate (beta-TCP) scaffold and permit cell proliferation during long-term incubation. The critical-size porous cylindrical scaffold (14 mm in diameter, 30 mm in length) with a central tunnel was impregnated with sheep mesenchymal stem cells. In the flow perfusion group, the hybrid scaffolds were continuously perfused with complete alpha-minimum essential medium via a peristaltic pump for 7, 14, and 28 days. In the static culture group, the hybrid composites were immersed in the medium without perfusion for 14 and 28 days. The daily glucose consumption was much higher in the flow perfusion group than in the static group (p < 0.001). In the flow perfusion group, glucose consumption increased dramatically in the first 14 days, and the increase slowed in the last 14 days. In the static group, the increase occurred only in the first 14 days. Cell viability via MTT colorimetry increased with time, which coincided with the results of glucose consumption. Histological study showed that the cells proliferated through the whole scaffolds under the flow perfusion culture. While under the static culture, the cells survived and proliferated only inside the first to third rows of the macropores under the scaffold surface. The cell quantity increased with time under flow perfusion culture. The results suggest that flow perfusion culture is superior to static culture for mesenchymal stem cell proliferation in the critical-size porous scaffold. This perfusion culture system permits a constant nutrition supply into the center of a large-scale scaffold for at least 4 weeks. Determination of D-glucose in the culture medium is a noninvasive way to survey cell proliferation in this system.