Design of experiments to assess the effect of culture parameters on the osteogenic differentiation of human adipose stromal cells

Abstract

Background Human adipose-derived stromal cells (hASCs) have been gaining increasing popularity in regenerative medicine thanks to their multipotency, ease of collection, and efficient culture. Similarly to other stromal cells, their function is particularly sensitive to the culture conditions, including the composition of the culture medium. Given the large number of parameters that can play a role in their specification, the rapid assessment would be beneficial to allow the optimization of their culture parameters. Method Herein we used the design of experiments (DOE) method to rapidly screen the influence and relevance of several culture parameters on the osteogenic differentiation of hASCs. Specifically, seven cell culture parameters were selected for this study based on a literature review. These parameters included the source of hASCs (the different providers having different methods for processing the cells prior to their external use), the source of serum (fetal bovine serum vs. human platelet lysate), and several soluble osteoinductive factors, including dexamethasone and a potent growth factor, the bone morphogenetic protein-9 (BMP-9). The expression of alkaline phosphatase was quantified as a readout for the osteogenic differentiation of hASCs. Results The DOE analysis enabled to classify the seven studied parameters according to their relative influence on the osteogenic differentiation of hASCs. Notably, the source of serum was found to have a major effect on the osteogenic differentiation of hASCs as well as their origin (different providers) and the presence of L-ascorbate-2-phosphate and BMP-9. Conclusion The DOE-based screening is a valuable approach for the classification of the impact of several cell culture parameters on the osteogenic differentiation of hASCs. Electronic supplementary material The online version of this article (10.1186/s13287-019-1333-7) contains supplementary material, which is available to authorized users.

Bone regeneration in rat calvarial defects using dissociated or spheroid mesenchymal stromal cells in scaffold-hydrogel constructs

“…Taken together, these findings suggest that the HPLG may have itself initiated the osteogenic differentiation of 2D and 3D BMSC (regardless of media supplements), thereby attenuating differences in gene expression, but promoting mineralization. Indeed, when used as a culture supplement, HPL promotes the osteogenic differentiation of MSC in vitro [ 54 – 56 ]. Moreover, several studies have reported positive effects of platelet-derived growth factors, e.g., PRP, on MSC osteogenic differentiation both in vitro and in vivo [ 23 , 57 – 60 ].…”

Continuous Electrical Stimulation Affects Initial Growth and Proliferation of Adipose-Derived Stem Cells

“…For this issue, in particular, adipose-derived stem cells are especially suitable as they are abundant and functional with an osteogenic potential [43][44][45]. The cells used in the present experiment were extracted and characterized as described previously [30,31]. Nevertheless, as the cells were received from three different healthy donors, a heterogenous population can be assumed and cell response to electrical stimulation may differ with regard to the donor age, the location of harvesting as well as the harvesting method.…”

“…For this purpose, the defect to be stimulated has to be filled with bone forming stem or progenitor cells in order to restore the missing bone volume. The success of osseous regeneration largely depends on the cell type and adipose-derived stem cells are commonly studied for bone tissue engineering approaches due to their multipotency, ease of the collection and efficient culture conditions [22,30]. Therefore, the aim of our present study was to analyze and establish suitable parameters for continuous electrical stimulation in order to improve the growth, viability and proliferation of human adipose-derived stem cells in vitro.…”

Critical Analysis of cGMP Large-Scale Expansion Process in Bioreactors of Human Induced Pluripotent Stem Cells in the Framework of Quality by Design

“…Although the DoEs and optimization through MVDA for the improvement of hiPSC culture conditions [44][45][46], downstream concentration [35], and differentiation [47][48][49] have been described, examples of employing a QRM and QbD approach for the optimization of hiPSC expansion are limited, likely because ATMPs, such as hiPSCs and their derivatives, are a relatively new category in drug regulation. Indeed, ATMPs were included in the pharmaceutical legislation only in 2009 [50].…”