Osteogenic Differentiation of Adipose-Derived Stromal Cells: From Bench to Clinics.

In addition to mesenchymal stem cells, adipose-derived stem/stromal cells (ASCs) are an attractive source for a large variety of cell-based therapies. One of their most important potential applications is related to the regeneration of bone tissue thanks to their capacity to differentiate in bone cells. However, this requires a proper control of their osteogenic differentiation, which depends not only on the initial characteristics of harvested cells but also on the conditions used for their culture. In this review, we first briefly describe the preclinical and clinical trials using ASCs for bone regeneration and present the quantitative parameters used to characterize the osteogenic differentiation of ASCs. We then focus on the soluble factors influencing the osteogenic differentiation of ACS, including the steroid hormones and various growth factors, notably the most osteoinductive ones, the bone morphogenetic proteins (BMPs). Impact statement Adipose-derived stromal/stem cells are reviewed for their use in bone regeneration.

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

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.

Solvent-free preparation of porous poly(l-lactide) microcarriers for cell culture.

Porous polymeric microcarriers are a versatile class of biomaterial constructs with extensive use in drug delivery, cell culture and tissue engineering. Currently, most methods for their production require potentially toxic organic solvents with complex setups which limit their suitability for biomedical applications and their large-scale production. Herein, we report an organic, solvent-free method for the fabrication of porous poly(l-lactide) (PLLA) microcarriers. The method is based on the spherulitic crystallization of PLLA in its miscible blends with poly(ethylene glycol) (PEG). It is shown that the PLLA spherulites are easily recovered as microcarriers from the blends by a water-based process. Independent control over microcarrier size and porosity is demonstrated, with a higher crystallization temperature leading to a larger size, and a higher PLLA content in the starting blend resulting in a lower microcarrier porosity. Microcarriers are shown to be biocompatible for the culture of murine myoblasts and human adipose stromal/stem cells (hASC). Moreover, they support not only the long-term proliferation of both cell types but also hASC differentiation toward osseous tissues. Furthermore, while no significant differences are observed during cell proliferation on microcarriers of two different porosities, microcarriers of lower porosity induce a stronger hASC osteogenic differentiation, as evidenced by higher ALP enzymatic activity and matrix mineralization. Consequently, the proposed organic-solvent-free method for the fabrication of biocompatible porous PLLA microcarriers represents an innovative methodology for ex vivo cell expansion and its application in stem cell therapy and tissue engineering. STATEMENT OF SIGNIFICANCE: We report a new solvent-free method for the preparation of porous polymeric microcarriers for cell culture, based on biocompatible poly(l-lactide), with independently controllable size and porosity. This approach, based on the spherulitic crystallization in polymer blends, offers the advantages of simple implementation, biological and environmental safety, easy adaptability and up-scalablility. The suitability of these microcarriers is demonstrated for long-term culture of both murine myoblasts and human adipose stromal/stem cells (hASCs). We show that prepared microcarriers support the osteogenic differentiation of hASCs, provided microcarriers of properly-tuned porosity are used. Hence, this new method is an important addition to the arsenal of microcarrier fabrication techniques, which will contribute to the adoption, regulatory approval and eventually clinical availability of microcarrier-based treatments and therapies.