GMP-compliant isolation and large-scale expansion of bone marrow-derived MSC.
BACKGROUND
Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components.


RESULTS
We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform.


CONCLUSIONS
The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.
Institut für Transfusionsmedizin, Universitätsklinikum Ulm, Ulm, Germany.
Natalie
N
Fekete
Markus T
MT
Rojewski
Daniel
D
Fürst
Ludwika
L
Kreja
Anita
A
Ignatius
Julia
J
Dausend
Hubert
H
Schrezenmeier
D000293:Adolescent; D000328:Adult; D001853:Bone Marrow / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D018925:Chemokines / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005947:Glucose / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism; D007621:Karyotyping / Q000379:methods; D019344:Lactic Acid / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D016133:Polymerase Chain Reaction / Q000379:methods; D013234:Stem Cells; D023822:Tissue Engineering / Q000379:methods
D018925:Chemokines; D003470:Culture Media; D016207:Cytokines; D016023:Integrins; D019344:Lactic Acid; D005947:Glucose