Effects of Flow Rate on Mesenchymal Stem Cell Oxygen Consumption Rates in 3D Bone-Tissue-Engineered Constructs Cultured in Perfusion Bioreactor Systems

Abstract

Bone grafts represent a multibillion-dollar industry, with over a million grafts occurring each year. Common graft types are associated with issues such as donor site morbidity in autologous grafts and immunological response in allogenic grafts. Bone-tissue-engineered constructs are a logical approach to combat the issues commonly encountered with these bone grafting techniques. When creating bone-tissue-engineered constructs, monitoring systems are required to determine construct characteristics, such as cellularity and cell type. This study aims to expand on the current predictive metrics for these characteristics, specifically analyzing the effects of media flow rate on oxygen uptake rates (OURs) of mesenchymal stem cells seeded on poly(L-lactic acid) (PLLA) scaffolds cultured in a flow perfusion bioreactor. To do this, oxygen consumption rates were measured for cell/scaffold constructs at varying flow rates ranging from 150 to 750 microliters per minute. Residence time analyses were performed for this bioreactor at these flow rates. Average observed oxygen uptake rates of stem cells in perfusion bioreactors were shown to increase with increased oxygen availability at higher flow rates. The residence time analysis helped identify potential pitfalls in current bioreactor designs, such as the presence of channeling. Furthermore, this analysis shows that oxygen uptake rates have a strong linear correlation with residence times of media in the bioreactor setup, where cells were seen to exhibit a maximum oxygen uptake rate of 3 picomoles O 2 /hr/cell.

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“…To overcome the diffusion limitation, various perfusion bioreactors have been developed over the years 6,10–18 . An example of a perfusion bioreactor is hollow fibre membrane bioreactors (HFMBs) 5,19,20 .…”

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“…Most probably, this is due to decreasing flow velocities (see Supplementary Figure 1). Supporting this hypothesis, Felder et al showed that the OC of mesenchymal stem cells decreases with lower flow rates (Felder et al, 2020[ 17 ]). Rennert et al demonstrated that higher perfusion rates lead to a higher total OC in a liver-on-a-chip platform (Rennert et al, 2015[ 42 ]).…”

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“…Indeed, the application of the millifluidic compartmentalization turned out to be a useful tool in examining both the h-TM cell behavior after exposure to hydrogen peroxide, as an oxidative stressor, and in enabling the analysis of the complex interactions between h-TM cells and their micro environmental components through an accurate and reproducible model. In fact, the limits of 3Dstatic systems are mainly related to a lack of oxygen availability [27], whereas the dynamic millifluidic platform provides a constant flow rate of the culture medium within the whole bioreactor circuit, allowing a continuous nutrient supply of oxygen to the connected multi-culture chambers, and thus overcoming not only the mass transport limitations, but also removing the metabolic products from the cells.…”