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Abstract
Dynamic culture protocols have recently emerged as part of (bone) tissue engineering strategies due to their ability to represent a more physiological cell environment in vitro. Here, we described how a perfusion flow induced by a simple bioreactor system improves proliferation and osteogenic commitment of human bone marrow stromal cells. L88/5 cells were cultured in poly(methyl methacrylate) custom-milled communicating well plates, in the presence of an osteogenic cocktail containing 1α,25-dihydroxyvitamin D3, L-ascorbic acid 2-phosphate, and β-glycerophosphate. The dynamic cell culture was maintained under perfusion flow stimulation at 1 mL/min for up to 4 days and compared with a static control condition. A cell viability assay showed that the proliferation associated with the dynamic cell culture was 20% higher vs. the static condition. A significantly higher upregulation of the osteogenic markers runt-related transcription factor 2 (RUNX2), collagen type I (COL1A1), osteocalcin (BGLAP), alkaline phosphatase (ALPL), and osteopontin (SPP1) was detected when the perfusion flow stimulation was administered to the cells treated with the osteogenic cocktail. An in silico analysis showed that in the dynamic cell culture condition (i) the shear stress in the proximity of the cell layer approximates 10−3 Pa, (ii) the nutrient and the waste product concentration is more homogeneously distributed than in the static counterpart, and (iii) perfusion flow was associated with higher nutrient consumption. In summary, increased cell proliferation and enhanced early phenotype commitment indicate that dynamic cell culture conditions, delivered via bioreactor systems, produce an enhanced in vitro environment for both basic and translational research in tissue engineering and regenerative medicine.
Highlights
Obtaining more physiological stem cell (SC) culture conditions to support in vitro cell expansion and/or to prompt a phenotype commitment of interest is a valuable objective for both basic and translational research
The ability to provide cell cultures with adequate nutrient supply and waste removal and to administer specific physical cues as differentiating signals would foster the study of molecular SC physiology and be relevant for the design of efficient protocols in tissue engineering (TE) and regenerative medicine (RM)
Traditional strategies for stem cell (SC) differentiation rely on soluble molecules, such as growth factors and cytokines, that induce a cascade of signalling pathways associated with phenotype commitment
Summary
Obtaining more physiological stem cell (SC) culture conditions to support in vitro cell expansion and/or to prompt a phenotype commitment of interest is a valuable objective for both basic and translational research. The main drawback in traditional static cell cultures is the lack of the sustained native diffusive exchanges This constraint might both affect the cell availability of glucose, an important nutrient for the generation of cellular energy, and consistently determine the accumulation of toxic metabolites such as lactate that could inhibit cell growth [1]. In this respect, including a perfusion flow would provide to the cells a more physiological environment [2, 3]. Besides increasing cell seeding and viability, fluid perfusion-induced shear stresses were demonstrated to drive osteogenic phenotype commitment [4–
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