Abstract
Introduction
Hematopoiesis takes place in the bone marrow niche. The niche is created by a specific interplay of different cell types and signalling molecules such as growth factors and cytokines. The self-renewal and survival of hematopoietic stem cells (HSCs) as well as their proliferation and differentiation are stimulated by these microenvironmental factors. Hematological disorders such as leukemia are associated with disruptions of the microenvironment within the bone marrow niche. To get further insights into the interplay of cells and molecules creating the niche and potential alterations due to hematological disorders, it is of importance to develop a model which mimics the niche in vitro. Thus, the aim of this study was to establish a 3D perfusion culture system for human mesenchymal stromal cells (MSCs), which constitute an important supportive cellular component of the bone marrow niche.
Methods
We used a modular perfusion culture system (Will W. Minuth, Regensburg, Germany). The system was already applied for culturing of a variety of different tissues, but not yet for culturing human MSCs.
MSCs were cultured on porous membrane filters consisting of mixed cellulose esters (pore size: 0.45 µm). Membranes were placed into a container, which was permanently perfused with fresh culture medium. Thus, the cells were constantly provided with nutrition while the accumulation of toxic metabolic products was prevented.
To establish the system, several parameters were varied in order to find out optimal conditions for the MSCs in perfusion culture. The influence of the following parameters was analyzed: the material of the membranes, the cell seeding volume, the position of the membranes in the perfusion culture container, the concentration of HEPES buffer in the medium and its flow rate. The viability of the MSCs in different culture conditions was tested by applying an MTS assay. Additionally, morphology of MSCs and the expression of exemplary selected genes important for the bone marrow niche (CXCL12 and JAG1) were analyzed. MSCs cultured under conventional cell culture conditions served as controls. Statistical analysis was performed by using the Student’s t-test (GraphPad Prism 6).
Results
We could show that the amount of viable MSCs cultured under ideal conditions in perfusion culture was considerably higher than in conventional cell cuture (OD450: 0.37 ± 0.03 vs. 0.2 ± 0.01, P-value < 0.01 %). A perfusion rate of 20.8 µL per minute and a HEPES concentration of 50 mM were observed to be optimal for the viability and growth of the MSCs. The cells showed no differences in gene expression levels due to the different culture conditions.
Table 1: Relative quantification of CXCL12 and JAG1 mRNA levels in MSCs DeltaCTCXCL12 DeltaCTJAG1 Perfusion culture 8.402 5.967 Conventional cell culture 8.008 6.750
Additionally, the MSCs cultured on membranes formed 3D-like networks. In perfusion culture, the MSCs seemed to grow in a more orderly manner compared to conventional cell culture conditions. No differences in morphology were observed due to the different culture conditions.
Discussion
The 3D perfusion culture system is sufficient to increase the viability and the growth of the MSCs without changing the gene expression profile of exemplary chosen genes relevant for homing and adhesion of HSCs in the bone marrow niche. The morphology of MSCs also did not change due to the different culture conditions.
Conclusion
This system can be used for further experiments including co-culturing experiments with MSCs and HSCs and/ or leukemic blasts and might be an important option to mimic the hematopoietic stem cell niche in vitro.
Disclosures
No relevant conflicts of interest to declare.
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