Mesenchymal Stem Cells - Basics and Clinical Application I by Birgit Weyand Massimo Dominici Ralf Hass Roland Jacobs & Cornelia Kasper

Author:Birgit Weyand, Massimo Dominici, Ralf Hass, Roland Jacobs & Cornelia Kasper
Language: eng
Format: epub
Publisher: Springer Berlin Heidelberg, Berlin, Heidelberg

5.2 Neurospheres Generation

The cellular pellet obtained from the digestion of adipose tissue is resuspended with proliferation medium [DMEM-HAM’s F12 (3:1), 10 % FBS, 1 % P/S, 20 ng/μL EGF, and 40 ng/μL FGF] and seeded at a density of 105 cells/cm2. Cells are cultured at 37 °C in humidified atmosphere with 5 % CO2. After 7 days under these culture conditions, most of the cells adhere to the tissue culture plastic, assuming a fibroblast like phenotype that is flattened and spindle shaped. Nevertheless, a small population of expanded cells is able to organize into spheres growing in suspension. These proliferating spheres, called “neurospheres,” are cell aggregates arranged in three-dimensional structures. It has been shown that the highest number of neurospheres from adipose tissue is generated in vitro when the culture medium is supplemented with serum and enriched with a combination of EGF and FGF. In addition, it is also necessary for the production of neurospheres that cells are cultured without any attachment factor [102, 109]). Once formed, floating neurospheres are passaged by harvesting and centrifugating the medium. Then, neurospheres are resuspended in proliferation medium and mechanically dissociated by vigorous trituration with a Pasteur pipette. At this point, cells are cultured at high density in new culture flasks with fresh proliferation medium for an additional 7 days.

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