Optimization of the cultivation conditions of mesenchymal stem cells with regard to media supplementation, the complexity of the cell construct, and the influence of oxygen

Abstract

Regenerative medicine aims to not only treat, but also cure diseases and pathological conditions. The utilization of human cells in cell-based therapies raises new hopes for achieving this, while at the same time bringing immense challenges for researchers, physicians and manufacturers. To reach efficient and reproducible treatment outcomes, it is essential to consider all steps along the fabrication of the cell-based product. Usually, the choice of cell sources, isolation methods, and injection modes are the major parameters studied here. However, in order to obtain fully functioning cell-based products and reliable tissue models, it is essential to devote research time to neglected aspects of the cell-based product chain, like optimization of in vitro cell cultivation protocols and the development of superior cultivation platforms. In this work, selected studies dedicated to the optimization and control of in vitro cultivation conditions are presented. Based on the example of clinically-relevant mesenchymal stem/stromal cells, the influence of three critical aspects ((i) media supplements, (ii) three-dimensional cultures and (ii) oxygen supply monitoring) on final product quality is demonstrated. In the first part, optimization of in vitro cell cultivation towards xeno-free conditions is performed. As a valuable alternative to the traditional media supplement fetal calf serum, the implementation of human platelet lysate was systematically evaluated in different culture systems. It was demonstrated that human platelet lysate is a xeno-free supplement, which outmatches the performance of fetal calf serum. A second major factor, the cultivation of cells in three-dimensional systems was also evaluated. In the next section of collected works, a tunable hydrogel-based platform for 3D cell cultivation of human mesenchymal stem cells was engineered and studied. Here, gelatin methacryloyl hydrogels with different degrees of functionalization, resulting in control over final mechanical properties, were synthetized and characterized considering their possible application as a 3D cultivation platform. Not only hydrogels of various uniform bulk mechanical properties could be created, but also techniques for creating stiffness gradient constructs from these hydrogels are reported in the presented works. In the third part, mesenchymal stem cells were modified with genetically encoded hypoxia biosensors and various two-dimensional and three-dimensional culture systems were evaluated with the help of these hypoxia-reporter cells. These hypoxia-reporter stromal cells are the first of their kind to be described in the literature, and allow the spatiotemporal recording of oxygen supply limitation in different in vitro systems. Taken together, the presented works demonstrate how so far less-considered factors like xeno-free media supplements, 3D cell cultivation in tunable hydrogels and genetically encoded reporter cells can all make a crucial contribution towards the improvement of cell-based products in regenerative medicine.