Open AccessPreparation of Anti‐Inflammatory Mesenchymal Stem/Precursor Cells (MSCs) Through Sphere Formation Using Hanging‐Drop Culture Technique
Author(s) -
Bartosh Thomas J.,
Ylostalo Joni H.
Publication year - 2014
Publication title -
current protocols in stem cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.658
H-Index - 28
eISSN - 1938-8969
pISSN - 1941-7322
DOI - 10.1002/9780470151808.sc02b06s28
Subject(s) - mesenchymal stem cell , microbiology and biotechnology , biology , paracrine signalling , lipopolysaccharide , stem cell , cell culture , immunology , biochemistry , genetics , receptor
Herein, we describe a protocol for preparation of pre‐activated anti‐inflammatory human mesenchymal stem/precursor cells (MSCs) in 3‐D culture without addition of exogenous chemicals or gene‐transfer approaches. MSCs are an easily procurable source of multipotent adult stem cells with therapeutic potential largely attributed to their paracrine regulation of inflammation and immunity. However, the culture conditions to prepare the ideal MSCs for cell therapy remain elusive. Furthermore, the reported lag time for activation in experimental models has prompted investigations on pre‐activating the cells prior to their administration. In this protocol, standard 2‐D culture‐expanded MSCs are activated by aggregation into 3‐D spheres using hanging‐drop cultures. MSC activation is evaluated by real‐time PCR and/or ELISA for anti‐inflammatory factors (TSG‐6, STC‐1, PGE2), and by a functional assay using lipopolysaccharide‐stimulated macrophage cultures. Further, we elucidate methods to prepare MSC‐sphere conditioned medium, intact spheres, and suspension of single cells from spheres for experimental and clinical applications. Curr. Protoc. Stem Cell Biol . 28:2B.06.1‐2B.06.23. © 2014 by John Wiley & Sons, Inc.