title: Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging-drop culture technique.
abstract: 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.
authors: Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White Hospital, Temple, Texas.
authors: Thomas J
authors: TJ
authors: Bartosh
authors: Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White Hospital, Temple, Texas.
authors: Joni H
authors: JH
authors: Ylostalo
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D046508:Culture Techniques / Q000379:methods*; D015232:Dinoprostone / Q000378:metabolism; D005615:Freezing; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D018874:Spheroids, Cellular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D015415:Biomarkers; D017077:Culture Media, Conditioned; D015232:Dinoprostone
keywords: LPS; MSC; activation; anti-inflammatory; macrophage; sphere; spheroid