Abstract
The therapeutic and differentiation potential of human mesenchymal stems cells (hMSCs) makes these cells a promising candidate for cellular therapies and tissue engineering. On the path of a successful medical application of hMSC, the cultivation of cells in a three-dimensional (3D) environment was a landmark for the transition from simple two-dimensional (2D) testing platforms to complex systems that mimic physiological in vivo conditions and can improve hMSC curative potential as well as survival after implantation. A 3D arrangement of cells can be mediated by scaffold materials where cells get entrapped in pores, or by the fabrication of spheroids, scaffold-free self-organized cell aggregates that express their own extracellular matrix. Independently from the cultivation method, cells expanded in 3D experience an inhomogeneous microenvironment. Many gradients in nutrient supply, oxygen supply, and waste disposal from one hand mimic in vivo microenvironment, but also put every cell in the 3D construct in a different context. Since oxygen concentration in spheroids is compromised in a size-dependent manner, it is crucial to have a closer insight on the thresholds of hypoxic response in such systems. In this work, we want to improve our understanding of oxygen availability and consequensing hypoxia onset in hMSC spheroids. Therefore, we utilized human adipose tissue-derived MSCs (hAD-MSCs) modified with a genetical sensor construct to reveal (I) the influence of spheroid production methods and (II) hMSCs cell number per spheroid to detect the onset of hypoxia in aggregates. We could demonstrate that not only higher cell numbers of MSCs, but also spheroid formation method plays a critical role in onset of hypoxia.
Highlights
Human mesenchymal stem cells are extensively studied in the field of regenerative medicine
We utilized human adipose tissue-derived MSCs modified with genetical sensor construct to reveal (I) the influence of spheroid production methods and (II) MSCs cell number per spheroid to detect the onset of hypoxia in aggregates
For the spheroid formation in well plates, a strong signal was obtained from a spheroids of 3 × 104 cells and larger (Figure 2A), for the hanging drop method the reporter protein expression starts at spheroid size from 7.5 × 104 and larger (Figure 2B)
Summary
Human mesenchymal stem cells (hMSC) are extensively studied in the field of regenerative medicine. Their ability of self-renewal and differentiation makes them a promising tool for biomedical applications (Dominici et al, 2006), hMSCs secrete cytokines and signal hormones which convey angiogenic and anti-apoptotic effects (Teixeira et al, 2013). Another promising bioregenerative potential of hMSCs comes from their ability to produce extracellular. In comparison to scaffoldbased platforms, which contain an exogenic compounds, cells arranged as spheroids build their own extracellular matrix to maintain their 3D organization
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