Hypoxia Onset in Mesenchymal Stem Cell Spheroids: Monitoring With Hypoxia Reporter Cells

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.

Modulation of Inherent Niches in 3D Multicellular MSC Spheroids Reconfigures Metabolism and Enhances Therapeutic Potential

“…In the present study, MSC spheroids that were assembled by 40,000 cells and cultivated for 2 days with a diameter of approximately 525 µm showed the best therapeutic potential. The discrimination between the investigated groups was thought to be attributed to the difference in the intrinsic gradients of oxygen, nutrients and signal molecules present in the spheroids [39,40]. Typically, as the spheroid size increases, the limited availability of oxygen owing to the increased diffusion distance is considered to mainly account for the development of a hypoxic core in spheroids, thereby altering cellular behaviors or even causing the death of interior cells [41][42][43].…”

Intravascular Application of Labelled Cell Spheroids: An Approach for Ischemic Peripheral Artery Disease

“…Even tough, from a clinical perspective, a substantial cell density is required for cell-based therapies to trigger tissue formation, the increasing length of nutrient transport by diffusion arises as a limitation. While Schmitz et al showed that MSC spheroids with diameters greater than 200 µm are vulnerable to hypoxia and implicitly cell death [33] several other studies inves-tigated the presence of a hypoxic core by means of histological approaches and indicated the presence of proliferating cells in spheroids up to 1000 µm in diameter [29,34,35]. It has to be borne in mind that in a clinical context, for peripheral arterial disease, the spheroidal size has to be subjected to further evaluation.…”

Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome

“…By establishing spheroid culture, more cells can be obtained than by monolayer cultures [182], therefore secreted factors amount increases together with an enhancement in anti-inflammatory factors production [183]. In stem cell spheroids the relative hypoxia located at the center of the sphere has a critical role [184] in the maintenance of stemness and for clinical application of MSCs [185]. Protein synthesis and cell volume increase can be uncoupled [186].…”