title: GMP-compliant isolation and expansion of bone marrow-derived MSCs in the closed, automated device quantum cell expansion system.
abstract: The estimated frequency of MSCs in BM is about 0.001-0.01% of total nucleated cells. Most commonly, one applied therapeutic cell dose is about 1-5 million MSCs/kg body weight, necessitating a reliable, fast, and safe expansion system. The limited availability of MSCs demands for an extensive ex vivo amplification step to accumulate sufficient cell numbers. Human platelet lysate (PL) has proven to be a safe and feasible alternative to animal-derived serum as supplement for MSC cultivation. We have investigated the functionally closed automated cell culture hollow fiber bioreactor Quantum cell expansion system as an alternative novel tool to conventional tissue flasks for efficient clinical-scale MSC isolation and expansion from bone marrow using PL. Cells expanded in the Quantum system fulfilled MSC criteria as shown by flow cytometry and adipogenic, chondrogenic, and osteogenic differentiation capacity. Cell surface expression of a variety of chemokine receptors, adhesion molecules, and additional MSC markers was monitored for several passages by flow cytometry. The levels of critical media components like glucose and lactate were analyzed. PDGF-AA, PDGF-AB/BB, bFGF, TGF-β1, sICAM-1, sVCAM-1, RANTES, GRO, VEGF, sCD40L, and IL-6 were assessed using a LUMINEX platform. Originally optimized for the use of fetal calf serum (FCS) as supplement and fibronectin as coating reagent, we succeeded to obtain an average of more than 100×10(6) of MSCs from as little as 18.8-28.6 ml of BM aspirate using PL. We obtained similar yields of MSCs/µl BM in the FCS-containing and the xenogen-free expansion system. The Quantum system reliably produces a cellular therapeutic dose in a functionally closed system that requires minimal manipulation. Both isolation and expansion are possible using FCS or PL as supplement. Coating of the hollow fibers of the bioreactor is mandatory when loading MSCs. Fibronectin, PL, and human plasma may serve as coating reagents.
authors.affiliation: Institut für Transfusionsmedizin, Universität Ulm, Ulm, Germany.
authors.forename: Markus T
authors.initials: MT
authors.lastname: Rojewski
authors.forename: Natalie
authors.initials: N
authors.lastname: Fekete
authors.forename: Stefano
authors.initials: S
authors.lastname: Baila
authors.forename: Kim
authors.initials: K
authors.lastname: Nguyen
authors.forename: Daniel
authors.initials: D
authors.lastname: Fürst
authors.forename: Delbert
authors.initials: D
authors.lastname: Antwiler
authors.forename: Julia
authors.initials: J
authors.lastname: Dausend
authors.forename: Ludwika
authors.initials: L
authors.lastname: Kreja
authors.forename: Anita
authors.initials: A
authors.lastname: Ignatius
authors.forename: Luc
authors.initials: L
authors.lastname: Sensebé
authors.forename: Hubert
authors.initials: H
authors.lastname: Schrezenmeier
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D019149:Bioreactors; D001792:Blood Platelets / Q000737:chemistry / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000592:standards*; D002454:Cell Differentiation; D002469:Cell Separation / Q000295:instrumentation*; D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007621:Karyotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype
chemical_list: D015415:Biomarkers; D016207:Cytokines; D005353:Fibronectins