A novel feature for monitoring the enzymatic harvesting process of adherent cell cultures based on lens-free imaging.

Adherent cell cultures are often dissociated from their culture vessel (and each other) through enzymatic harvesting, where the detachment response is monitored by an operator. However, this approach is lacking standardisation and reproducibility, and prolonged exposure or too high concentrations can affect the cell's viability and differentiation potential. Quantitative monitoring systems are required to characterise the cell detachment response and objectively determine the optimal time-point to inhibit the enzymatic reaction. State-of-the-art methodologies rely on bulky imaging systems and/or features (e.g. circularity) that lack robustness. In this study, lens-free imaging (LFI) technology was used to develop a novel cell detachment feature. Seven different donors were cultured and subsequently harvested with a (diluted) enzymatic harvesting solution after 3, 5 and 7 days of culture. Cell detachment was captured with the LFI set-up over a period of 20 min (every 20 s) and by optimising the reconstruction of the LFI intensity images, a new feature could be identified. Bright regions in the intensity image were identified as detaching cells and using image analysis, a method was developed to automatically extract this feature, defined as the percentage of detached cell regions. Next, the method was quantitatively and qualitatively validated on a diverse set of images. Average absolute error values of 1.49%, 1.34% and 1.97% were obtained for medium to high density and overconfluent cultures, respectively. The detachment response was quantified for all conditions and the optimal time for enzyme inhibition was reached when approximately 92.5% of the cells were detached. On average, inhibition times of 9.6-11.1 and 16.2-17.2 min were obtained for medium to high density and overconfluent cultures, respectively. In general, overconfluent cultures detached much slower, while their detachment rate was also decreased by the diluted harvesting solution. Moreover, several donors exhibited similar trends in cell detachment behaviour, with two clear outliers. Using the novel feature, measurements can be performed with an increased robustness, while the compact LFI design could pave the way for in situ monitoring in a variety of culture vessels, including bioreactors.

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

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.

The combined mechanism of bone morphogenetic protein- and calcium phosphate-induced skeletal tissue formation by human periosteum derived cells.

When combining osteogenic progenitor cells such as human periosteum derived cells (hPDCs) with osteoconductive biomaterials like calcium phosphate (CaP)-scaffolds, in vivo bone formation can be achieved. This process is dependent on the early activation of Bone morphogenetic protein (BMP)-signalling. However, the bone forming process is slow and routinely only a limited amount of bone and bone marrow is formed. Therefore, we hypothesised that a robust clinically relevant outcome could be achieved by adding more physiological levels of potent BMP-ligands to these cell- and CaP-based constructs. For this, hPDCs were characterised for their responsiveness to BMP-ligands upon in vitro 2D stimulation. BMP-2, -4, -6 and -9 robustly induced osteochondrogenic differentiation. Subsequently, these ligands were coated onto clinically approved CaP-scaffolds, BioOss® and CopiOs®, followed by hPDC-seeding. Protein lysates and conditioned media were investigated for activation of BMP signalling pathways. Upon in vivo implantation, the most abundant bone formation was found in BMP-2 and BMP-6-coated scaffolds. Implanted cells actively contributed to the newly formed bone. Remnants of cartilage could be observed in BMP-coated CopiOs®-constructs. Computational analysis displayed that the type of BMP-ligand as well as the CaP-scaffold affects skeletal tissue formation, observed in a qualitative as well as quantitative manner. Furthermore, the in vitro mechanism appears to predict the in vivo outcome. This study presents further evidence for the potential of BMP-technology in the development of clinically relevant cell-based constructs for bone regenerative strategies.