Human periosteal-derived cell expansion in a perfusion bioreactor system: proliferation, differentiation and extracellular matrix formation

Abstract

Perfusion bioreactor systems have shown to be a valuable tool for the in vitro development of three-dimensional (3D) cell-carrier constructs. Their use for cell expansion, however, has been much less explored. Since maintenance of the initial cell phenotype is essential in this process, it is imperative to obtain insight into the bioreactor-related variables determining cell fate. Therefore, this study investigated the influence of fluid flow-induced shear stress on the proliferation, differentiation and matrix deposition of human periosteal-derived cells in the absence of additional differentiation-inducing stimuli; 120 000 cells were seeded on additive manufactured 3D Ti6Al4V scaffolds and cultured for up to 28 days at different flow rates in the range 0.04-6 ml/min. DNA measurements showed, on average, a three-fold increase in cell content for all perfused conditions in comparison to static controls, whereas the magnitude of the flow rate did not have an influence. Contrast-enhanced nanofocus X-ray computed tomography showed substantial formation of an engineered neotissue in all perfused conditions, resulting in a filling (up to 70%) of the total internal void volume, and no flow rate-dependent differences were observed. The expression of key osteogenic markers, such as RunX2, OCN, OPN and Col1, did not show any significant changes in comparison to static controls after 28 days of culture, with the exception of OSX at high flow rates. We therefore concluded that, in the absence of additional osteogenic stimuli, the investigated perfusion conditions increased cell proliferation but did not significantly enhance osteogenic differentiation, thus allowing for this process to be used for cell expansion. Copyright © 2014 John Wiley & Sons, Ltd.

Quantitative Validation of the Presto Blue™ Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System

“…Additionally, the conversion rates determined for the different conditions were not influenced by the perfusion velocity (Fig. 6B) indicating that, next to the proliferation, the PB conversion rates and therefore the metabolic activity of the cell population was not influenced by flow rate within the examined range corresponding with initial shear stress values between 7E-3 and 3.08E-2 Pa. 37 Although flow rate does not influence the conversion rate of the PB assay, it should be high enough to prevent local saturation of the assay at the outlet of the scaffold. To determine at which combinations of flow rate and cell number this could influence the readouts, mass balance equations were used to determine the relative saturation of the linear PB operating window in the medium reservoir and at the scaffold inlet and outlet in functions of flow rate, cell number, measurement volume ,and incubation time.…”

“…13,34,36,37 Forty-five minutes after seeding, 60 mL of culture medium was added and 135 min later the medium volume was topped up to 1 mL. Scaffolds were statically incubated overnight in standard culture conditions (37°C, 5% CO 2 , 95% relative humidity).…”

“…Since the seeding process resulted in homogenous and reproducible seeding efficiencies in previous work (*60%), this was not assessed separately in this work. 34,36,37 To determine the quantitative correlation between the PB signal and the cell content in a 3D static culturing system, scaffolds were transferred to 12-well plates containing 3 mL of culture medium after seeding. The medium was refreshed every 2 days and PB measurements were performed on days 1, 6, 10, 15, and 22 by replacing the culture medium with 3 mL of the PB solution and incubating the scaffolds in standard cell culture conditions for 3 h. The PB signal was measured in triplicate as described before.…”

“…34,36,37 Scaffolds were subsequently press-fitted in an in-house developed bioreactor chamber 13,34,36,37 ensuring forced perfusion through the scaffold and cultured at a flow rate of 1 mL/min in a nonhumidified incubator (37°C, 5% CO 2 ). The total medium volume in each circuit was 10 mL, of which 7.2 mL was located in the medium reservoir.…”

“…The green area shows the measurement conditions resulting in values within the linear operating window of the assay. Color images available online at www.liebertpub.com/tec in nutrient diffusion, 34,37,[39][40][41] the difference in the PB conversion rate might be related to limited diffusion.…”