title: Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum.
abstract: Adult mesenchymal stem cells (MSCs) are considered as valuable mediators for tissue regeneration and cellular therapy. This study was performed to develop conditions for regularly propagating a clinical quantity of > 2 x 10(8) MSCs without animal serum from small bone marrow (BM) aspiration volumes within short time. We established optimized culture conditions with pooled human platelet lysate (pHPL) replacing fetal bovine serum (FBS) for MSC propagation. MSC quality, identity, purity, and function were assessed accordingly. Biologic safety was determined by bacterial/fungal/mycoplasma/endotoxin testing and genomic stability by array comparative genomic hybridization (CGH). We demonstrate that unmanipulated BM can be used to efficiently initiate MSC cultures without the need for cell separation. Just diluting 1.5-5 mL heparinized BM per 500 mL minimum essential medium supplemented with L-glutamine, heparin, and 10% pHPL sufficiently supported the safe propagation of 7.8 +/- 1.5 x 10(8) MSCs within a single 11- to 16-day primary culture under defined conditions. This procedure also resulted in sustained MSC colony recovery. MSC purity, immune phenotype, and in vitro differentiation potential fully matched current criteria. Despite high proliferation rate, MSCs showed genomic stability in array CGH. This easy single-phase culture procedure can build the basis for standardized manufacturing of MSC-based therapeutics under animal serum-free conditions for dose-escalated cellular therapy and tissue engineering.
authors.affiliation: Stem Cell Research Unit, Medical University of Graz, Graz, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schallmoser
authors.forename: Eva
authors.initials: E
authors.lastname: Rohde
authors.forename: Andreas
authors.initials: A
authors.lastname: Reinisch
authors.forename: Christina
authors.initials: C
authors.lastname: Bartmann
authors.forename: Daniela
authors.initials: D
authors.lastname: Thaler
authors.forename: Camilla
authors.initials: C
authors.lastname: Drexler
authors.forename: Anna C
authors.initials: AC
authors.lastname: Obenauf
authors.forename: Gerhard
authors.initials: G
authors.lastname: Lanzer
authors.forename: Werner
authors.initials: W
authors.lastname: Linkesch
authors.forename: Dirk
authors.initials: D
authors.lastname: Strunk
mesh_terms: D000293:Adolescent; D000328:Adult; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D003470:Culture Media; D016895:Culture Media, Serum-Free / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008875:Middle Aged; D009693:Nucleic Acid Hybridization; D013234:Stem Cells; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D003470:Culture Media; D016895:Culture Media, Serum-Free